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GLOBAL DISTRIBUTIONS AND NATURAL SOURCES OF BROMINATED
VERY SHORT-LIVED SUBSTANCES
A Dissertation
by
YINA LIU
Submitted to the Office of Graduate Studies of Texas A&M University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Chair of Committee, Shari A. Yvon-Lewis Co-Chair of Committee, Daniel C.O. Thornton Committee Members, Thomas S. Bianchi Lisa Campbell John D. Kessler Gunnar W. Schade Head of Department, Piers Chapman
August 2013
Major Subject: Oceanography
Copyright 2013 Yina Liu
ii
ABSTRACT
Brominated very short-lived substances (BrVSLS) are atmospherically
important trace gases that play an important role in stratospheric ozone destruction.
Major BrVSLS including bromoform (CHBr3), dibromomethane (CH2Br2),
chlorodibromomethane (CHClBr2), and bromodichloromethane (CHBrCl2) are thought
to be predominately formed naturally via vanadium bromoperoxidase (V-BrPO)
mediated halogenation of organic matter (OM). The objective of this research was to
couple field observations and laboratory experiments to understand global
distributions, saturation anomalies, fluxes, and identify natural sources of BrVSLS.
All the trace gases were measured with gas chromatography mass spectrometry (GC-
MS).
Field observations were conducted in the Pacific and Atlantic Oceans. Results
from field observations showed that BrVSLS tend to be elevated in biologically active
waters, such as coastal waters, the productive surface open ocean, and at chlorophyll
maximum depths. The production of natural BrVSLS is likely controlled by complex
biogeochemical factors in the ecosystems. CH2Br2 was thought to be derived from the
same source(s) as CHBr3, but results presented in this dissertation suggest they may in
fact be derived from disparate sources.
Screening for important BrVSLS producers was attempted in the laboratory.
Only 2 out of 9 phytoplankton species screened show observable BrVSLS production.
CH2Br2 production was only observed in 1 species screened. Chloroperoxidase-like
iii
activity in diatom was observed for the first time, which provided evidence for
biological production of chloroform (CHCl3).
The role of dissolved organic matter (DOM) in controlling BrVSLS production
was investigated in the laboratory. Production of BrVSLS varied significantly with
different model DOM compounds upon V-BrPO mediated halogenation. Certain
DOM enhanced BrVSLS production, but the majority of the model DOM compounds
tested in this study either interfered with or had no observable effect on BrVSLS
production. Further evidences showed that V-BrPO mediated halogenation can alter
DOM chemical characteristics. Alteration of colored dissolved organic matter
(CDOM) in terms of “bio-bleaching” was observed in model lignin phenol compounds
and CDOM collected from two cyanobacterial cultures.
Results from this study suggest that the presence of V-BrPO producing
phytoplankton is essential for enhanced BrVSLS production, as V-BrPO induced
brominated reactive species, such as hypobromous acid (HOBrenz), is required.
However, BrVSLS production rates are largely controlled by other biogeochemical
factors in seawater, such as DOM composition. Results from this study also suggest
that V-BrPO activity not only plays an essential role in BrVSLS production, but it also
plays a significant role in the transformation of DOM and may be a significant
component of the marine carbon cycle.
iv
ACKNOWLEDGEMENTS
I am especially grateful for having Drs. Shari Yvon-Lewis and Daniel
Thornton as my advisers. Without them, this research would not be possible. I
especially thank Shari for her support and allowing me to learn different skills from
other labs. Her support is essential for such an interdisciplinary research. I thank Dan,
who not only being a great adviser, but his insight also broadens my horizon of
thinking. I would like to thank my committee members, Drs. Thomas Bianchi, Lisa
Campbell, John Kessler, and Gunnar Schade, for their guidance and support
throughout the course of this research.
I thank Dr. Steven Manley at California State University, Long Beach, who
generously donated vanadium bromoperoxidases that were used in part of this study. I
also thank his insight; which helped developed some of the hypotheses in my research.
I sincerely thank Drs. François Primeau, Susan Trumbore, and Ellen Druffel at
University of California, Irvine, for their guidance during my undergraduate
researches. They not only provided me with opportunities to expose to research as an
undergraduate student, the experiences gained when working with them also
motivated me to pursued scientific research as my lifetime career.
I thank Drs. James Butler, Lei Hu, Joseph Salisbury, Richard Smith, and Julia
O’Hern, for their contributions to my publications (Chapters II, II, and IV). Thanks
also go to the department faculty and staff for making my time at Texas A&M
University a great experience.
v
I thank my friends in College Station, Fenix, Jan, Ruifang, Brad, Eric, and
Mike. I especially thank Fenix, who is being a great labmate and colleague. I thank my
friends back in California, UK, and China, Yu, Shanlin, Mo, Hai-lun, Ka-Ki, Ka-Yan,
Yuan-yuan, Wen-jun, and Yan-na. Their friendships and support mean a lot to me.
I thank my parents for their love and unconditional support for my whole life.
Without their love, nothing is possible. I thank my other family members, especially
my aunt and uncle, who took care of me in California. I would like to thank my
cousins for their company back in California, especially Wei-qi.
This research is partially supported by National Oceanic and Atmospheric
Administration (NOAA) grant NA06OAR4310049 and National Science Foundation
(NSF) grant OCE 0927874 to S.A. Y-L.
vi
NOMENCLATURE
BrVSLS Brominated very short-lived substances
Bry Inorganic bromine
CDOM Colored dissolved organic matter
CHBr3 Bromoform
CH2Br2 Dibromomethane
CHClBr2 Chlorodibromomethane
CHBrCl2 Bromodichloromethane
CO Carbon monoxide
CO2 Carbon dioxide
COS Carbonyl sulfide
DOC Dissolved organic carbon
DOM Dissolved organic matter
H2O2 Hydrogen peroxide
HAA Haloacetic acid
ODSs Ozone depleting substances
OM Organic matter
ppt Parts per trillion
patm Pico atmosphere
PGI Product gas injection
SGI Source gas injection
V-BrPO Vanadium bromoperoxidase
vii
TABLE OF CONTENTS
Page
ABSTRACT ................................................................................................................... ii
ACKNOWLEDGEMENTS .......................................................................................... iv
NOMENCLATURE ...................................................................................................... vi
TABLE OF CONTENTS ............................................................................................. vii
LIST OF FIGURES ...................................................................................................... ix
LIST OF TABLES ....................................................................................................... xv
CHAPTER I INTRODUCTION ................................................................................... 1
CHAPTER II CHBR3, CH2BR2 AND CHCLBR2 IN U.S. COASTAL WATERS DURING THE GULF OF MEXICO AND EAST COAST CARBON (GOMECC) CRUISE ......................................................................................................................... 6
2.1 Introduction .......................................................................................................... 6 2.2 Methods ................................................................................................................ 8 2.3 Results and discussions ...................................................................................... 11 2.4 Conclusion ......................................................................................................... 29
CHAPTER III SPATIAL DISTRIBUTION OF BROMINATED VERY SHORT-LIVED SUBSTANCES IN THE EASTERN PACIFIC .............................................. 32
3.1 Introduction ........................................................................................................ 32 3.2 Sampling and analyses methods ........................................................................ 35 3.3 Results and discussion ....................................................................................... 40 3.4 Conclusions ........................................................................................................ 60
CHAPTER IV SPATIAL AND TEMPORAL DISTRIBUTIONS OF BROMOFORM AND DIBROMOMETHANE IN THE ATLANTIC OCEAN AND THEIR RELATIONSHIP WITH PHOTOSYNTHETIC BIOMASS ................ 62
4.1 Introduction ........................................................................................................ 62 4.2 Method ............................................................................................................... 67 4.3 Results and discussion ....................................................................................... 74 4.4 Conclusion ....................................................................................................... 103
viii
CHAPTER V MARINE DISSOLVED ORGANIC MATTER (DOM) COMPOSITION DRIVES THE PRODUCTION AND CHEMICAL SPECIATION OF BROMINATED VERY SHORT-LIVED SUBSTANCES ................................. 105
5.1 Introduction ...................................................................................................... 105 5.2 Methods ............................................................................................................ 108 5.3 Results .............................................................................................................. 116 5.4 Discussion ........................................................................................................ 127 5.5 Conclusion ....................................................................................................... 138
CHAPTER VI PRODUCTION OF BROMINATED VERY SHORT-LIVED SUBSTANCES (BRVSLS) BY PHYTOPLANKTON ............................................. 140
6.1 Introduction ...................................................................................................... 140 6.2 Methods ............................................................................................................ 142 6.3 Results .............................................................................................................. 152 6.4 Discussion ........................................................................................................ 157 6.5 Conclusion ....................................................................................................... 162
CHAPTER VII EFFECTS OF VANADIUM BROMOPEROXIDASE HALOGENATION ON DISSOLVED ORGANIC MATTER AND IMPLICATIONS FOR THE MARINE CARBON CYCLE ..................................... 163
7.1 Introduction ...................................................................................................... 163 7.2 Method ............................................................................................................. 166 7.3 Results .............................................................................................................. 168 7.4 Discussion ........................................................................................................ 172 7.5 Conclusion ....................................................................................................... 175
CHAPTER VIII CONCLUSIONS ............................................................................. 177
8.1 Result summary ................................................................................................ 177 8.2 An outlook from a conceptual model ............................................................... 179 8.3 Conclusions and future works .......................................................................... 181
REFERENCES ........................................................................................................... 183
APPENDIX ................................................................................................................ 212
ix
LIST OF FIGURES
Page
Figure 1-1. Brominated organic matter in marine and atmospheric environments derived from a conceptual model of V-BrPO enzyme-mediated BrVSLS formation mechanisms. Marine algae that produce V-BrPO can oxidize bromine to form reactive species such as hypobromous acid (HOBr), in the presence of H2O2. HOBr can react with a wide variety of DOM compounds and consequently form brominated DOM. Some brominated DOM may release BrVSLS and contribute to catalytic ozone destruction reactions; but some brominated DOM maybe stable enough and can potentially be sorbed to sinking particulates; hence being transported into deeper waters. Some of the brominated OM on the particulates may be stable enough to resist degradations in the water column and in the sediments. Atmospheric interactions shown in this figure is adapted from Salawitch [2006]. ................................................................................................ 5
Figure 2-1. Map showing the Gulf of Mexico and East Coast Carbon (GOMECC) cruise with track (▬), 200m isobath (▬), yearday (●), treated water outfalls (♦), and seawater-cooled nuclear power plants (♦). ............................... 9
Figure 2-2. Time series of salinity (▬) and sea-surface temperature (▬) (a), chlorophyll a concentrations (▬) (b), CDOM concentrations (▬) (c), and wind speed (▬) (d) for the GOMECC cruise. The gray shadings highlight open ocean data. ............................................................................................... 12
Figure 2-3. Daily 48 hr 100m above sea surface Hysplit air mass back trajectories for selected points (▬) along the GOMECC cruise track (▬), and back trajectories for selected points with negative saturation anomalies (YD 199 – 200) (▬). (★) indicated the starting points of the back trajectories. ..... 13
Figure 2-4. Time series of atmospheric mixing ratios (a) and water concentrations (b) for CHBr3 (□), CH2Br2 (+), and CHClBr2 (×).The gray shadings highlight open ocean data. ................................................................................ 19
Figure 2-5. Time series of saturation anomalies (a), fluxes (b), and calculated production (c) for CHBr3 (□), CH2Br2 (+), and CHClBr2 (×).The gray shadings highlight open ocean data. ................................................................. 25
Figure 3-1. Sampling station map for the Halocarbon Air-Sea Transect – Pacific cruise, superimposed on March – April 2010 monthly averaged SeaWiFs chlorophyll a concentrations [Acker and Leptoukh, 2007]. ............................. 35
Figure 3-2. Salinity profile for the HalocAST-P cruise. .................................................. 42
x
Figure 3-3. Temperature-salinity (TS) diagram. (a) Below ~ 500 m of casts 4 (red), 5 (green), 6 (blue), and 7 (magenta) were the Antarctic intermediate water (AAIW; marked with black square). Casts 2 and 8 were plotted in grey lines as reference of water masses not in the AAIW. (b) Below ~300 m of casts 21(red), 22 (green), 23 (blue), and 24 (magenta) were the North Pacific intermediate water (NPIW; marked with black square). Casts 22 and 25 (TS only, no BrVSLS data collected for Cast 25, conducted in Puget Sound, Seattle, USA) were plotted in grey lines as reference of water masses not in the NPIW. ......................................................................... 43
Figure 3-4. (a) Dissolved oxygen profile and (b) CFC-11 profile during the HalocAST-P cruise. .......................................................................................... 44
Figure 3-5. (a) Silicate (HSiO3-), (b) orthophosphate (HPO4
2-), and (c) nitrate (NO3-
) profile during the HalocAST-P cruise. ........................................................... 45
Figure 3-6. Latitudinal distributions of mean mixed layer CHBr3, CH2Br2, CHClBr2, and CHBrCl2 concentrations, error bars indicate ±1 standard deviation of the mixed layer concentrations. .................................................... 48
Figure 3-7. Depth profiles of (a) CHBr3, (b) CH2Br2, (c) CHClBr2, (d) CHBrCl2 and (e) CHCl3. White line marks the bottom of mixed layer, black line marks the depths of chlorophyll a maxima, and red line marks the bottom of the euphotic zone. ................................................................................................... 49
Figure 3-8. Boxplots of data grouped based on geographical setting (open ocean vs. coastal ocean), and data grouped based on water column layers (mixed layer, below mixed layer within euphotic zone, and below euphotic zone), for CHBr3 (a and b), CH2Br2 (c and d), CHClBr2 (e and f), and CHBrCl2 (g and h). Horizontal line in the box indicates median of data, filled square indicates mean of data, box range indicates 25th to 75th percentile of data, whisker indicates 10th to 90th percentile of data, open box indicates 5th to 95th percentile of data. Stars indicate minimum and maximum of data. Note that the y-axis is a log-scale. ...................................... 50
Figure 4-1. Ship tracks for the GasEx98 (red line), BLAST-II (grey line), A16N (magenta line), A16S (blue line), and HalocAST-A (green line) cruises. ....... 66
Figure 4-2. 24 hours averaged wind speed at 10 m above sea surface (u10) for (a) the BLAST-II, A16N, A16S, and HalocAST-A cruises and (b) GasEx98 Legs 1, 2, 3, and 4. ............................................................................................ 71
Figure 4-3. Latitudinal distributions of CHBr3 and CH2Br2 atmospheric mixing ratios (a and c) and seawater concentrations (b and d) measured during the BLAST-II, A16N, A16S, and HalocAST-A cruises. ................................. 76
xi
Figure 4-4. Longitudinal distributions of CHBr3 and CH2Br2 atmospheric mixing ratios (a and c) and seawater concentrations (b and d) measured during the GasEx98 cruise Leg 1 and GasEx98 Legs 2 to 4. ...................................... 79
Figure 4-5. Data deviation from individual cruise mean of CHBr3 during the GasEx98 leg 1 (a), legs 2 to 4 (b), A16N (c), A16S (d), BLAST-II (e), and HalocAST-A (f) cruises, superimpose on 9-km resolution monthly averaged chlorophyll a concentration observed during the time of the cruise from SeaWiFS, except for the BLAST-II cruise, for which monthly climatology was used (NASA Giovanni [Acker and Leptoukh, 2007]). Time frame over which the data were acquired is labeled on each plot. Red data points indicate positive deviation and grey data points indicate negative deviation. Color bar indicates chlorophyll a concentration. .............. 82
Figure 4-6. Latitudinal distributions of CHBr3 and CH2Br2 saturation anomalies (Δ) (a and c) and fluxes (b and d) calculated for the BLAST-II, A16N, A16S, and HalocAST-A cruises. ................................................................................. 84
Figure 4-7. Longitudinal distributions of CHBr3 and CH2Br2 saturation anomalies (Δ) (a and c) and fluxes (b and d) calculated for the GasEx98 cruise Leg 1 and Legs 2 to 4. ................................................................................................ 85
Figure 4-8. Boxplots of data from the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises in different geographical regimes (a and c). Due to the scarcity of coastal data from these cruises (n = 6), coastal data collected from the Gulf of Mexico and East Coast Carbon (GOMECC) cruise [Liu et al., 2011] were added to the coastal data when creating the boxplot and serve as a reference for representative comparison (grey shaded box). The open ocean regime is further divided into regimes with low ([chl a] < 0.1), medium (0.1 ≤ [chl a] < 0.2), and high ([chl a] ≥ 0.2) chlorophyll a concentrations (b and d). Line in the box indicates median of data, filled square indicates mean of data, box range indicates 25th to 75th percentile of data, whisker indicates 10th to 90th percentile of data, open box indicates 5th to 95th percentile of data. Stars indicate minimum and maximum of data. ............................................................................................. 91
Figure 4-9. Scatter plots of CHBr3 (a) and CH2Br2 (b) concentrations vs. chlorophyll a concentration, for the BLAST-II, GasEx98, A16N, A16S, and HalocAST-A cruises. ................................................................................. 95
Figure 4-10. Scatter plots of CHBr3 seawater concentration vs. CH2Br2 seawater concentration for the BLAST-II, GasEx98, A16N, A16S, and HalocAST-A cruises. ........................................................................................................ 103
Figure 5-1. BrVSLS concentrations of (a) CHBr3, (b) CHClBr2, and (c) CHBrCl2, measured from HOBrenz halogenation of a series of model DOM compounds. Grey bar chart shows BrVSLS concentration resulted from
xii
different batches of ASW treated with different batches of V-BrPO. Filled circles are mean concentrations of BrVSLS produced by each individual model DOM compound, with error bar of ± 1 standard deviation, plotting on top of their corresponding batch of ASW halogenated by their corresponding batch of V-BrPO. DOM group labeled red indicates BrVSLS production was significantly less than V-BrPO treated ASW without model DOM added (interfered). DOM group labeled black indicates BrVSLS production was not significantly different from V-BrPO treated ASW without model DOM added (no observable effect). DOM group labeled green indicated BrVSLS production was significantly higher than V-BrPO treated ASW without model DOM added (enhanced). ........................................................................................... 119
Figure 5-2. Scatter plot of CHBr3 concentration vs. CHClBr2 and CHBrCl2 concentrations for (a) single DOM model compound experiment and (b) dual DOM model compounds experiment. Blue data points presented CHBr3 concentration vs. CHClBr2 concentration. Red data points presented CHBr3 concentration vs. CHBrCl2 concentration. In panel (b), Filled squares are data observed from tryptophan mixed with urea. Filled triangles are data observed from vanillin mixed with urea. Stars are data observed from phenol red mixed with urea. Open up-side-down triangles are data observed from D-glucose mixed with urea. Open circles are data observed from ultra-pure water mixed with urea. .......................................... 121
Figure 5-3. Brominated trihalomethane speciation across the model DOM compounds examined, and in comparison with pre- and post- V-BrPO treated artificial seawater (ASW and HASW, respectively) and aged Gulf of Mexico seawater (GoMSW and HGoMSW, respectively). ....................... 122
Figure 5-4. CHBr3 concentrations observed from dual model DOM compound experiment. (a) Tryptophan, (b) vanillin, (c) phenol red, (d) D-glucose, and (e) ultra-pure water were mixed with urea at relative proportions of 50%, 10%, 0.5%, and 0% (i.e. 100% urea), to make up a total DOM concentration of ~1000 µM. Grey bar chart plots CHBr3 concentration observed from V-BrPO treated ASW. ............................................................ 125
Figure 5-5. Brominated trihalomethane speciation at mixing proportion of 50%, 10%, 0.5%, and 0% of DOMinterfered and DOMno_effect with urea: (a) Tryptophan, (b) vanillin, (c) phenol red, (d) D-glucose, and (e) ultra-pure water. .............................................................................................................. 126
Figure 5-6. Absorbance of phenol red (λ = 433) and bromophenol blue (λ = 592) before (initial) and after (final) V-BrPO treatment of 10% phenol red + 90% urea samples. .......................................................................................... 134
Figure 5-7. Brominated trihalomethane speciation observed during the Halocarbon Air-sea Transect – Atlantic (HalocAST-A) cruise. ........................................ 136
xiii
Figure 6-1. Map of locations where the stock cultures were isolated from. Information was provided by the National Center of Marine Algae and Microbiota (NCMA). Orange symbols indicate diatom species, magenta symbol indicates cryptophte species, purple symbol indicates dinoflagellate species, grey symbol indicates prasinophyte species, green symbol indicates open ocean cyanobacteria species, and pink symbol indicates coastal cyanobacteria species. Stars indicate species with BrVSLS production. ....................................................................................... 144
Figure 6-2. Schematic of incubation vessel used for phytoplankton culture screening. Any trace gases produced by the phytoplankton were equilibrated with the headspace. Port A was connected to the GC-MS system to analyze for halocarbon production. A 0.2µm air filter was connected to port B, which served as makeup gas inflow to compensate pressure change inside the vessel during halocarbon headspace sampling. ... 146
Figure 6-3. Schematic of light transmission method, used for assessing cell density. A full spectrum light source was place at one side of the incubation vessel. A LiCor LI-250A light meter was place inline on the other side of the incubation vessel to measure light transmission through the incubation vessel. ............................................................................................................. 148
Figure 6-4. An example of cell density (Achnanthes longipes Agardh), presented in light transmission (Itransmission, µmol photon m-2 s-1), as a function of time. Trace gas samples were analyzed once the cell cultures reached stationary phase. .............................................................................................................. 149
Figure 6-5. Amounts (in aqueous concentration pmol L-1) of CHBr3, CH2Br2, CHClBr2, CHBrCl2, and CHCl3 produced by A. longipes and Synechococcus sp. in cultures of different ages. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater. ............................................. 154
Figure 6-6. Relative abundances of CHBr3, CHClBr2, and CHBrCl2 in the brominated trihalomethane (BTHM) pool in A. longipes (A.I.) and Synechococcus sp. (Syn) cultures. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater. ................................................................ 155
Figure 6-7. Relative abundances of CHBr3, CHClBr2, CHBrCl2, and CHCl3 in the trihalomethane (THM) pool in three different batches of A. longipes (A.I.) cultures. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater. ............................................................................................ 156
xiv
Figure 7-1. Fluorescence of V-BrPO treated (a) vanillin (red), (b) syringaldehyde (blue), and (c) ferulic acid (black) and their controls (grey) as a function of time. Error bars indicate 1 standard deviation (± 1σ) of replicates, number of samples (n) were 3 and 2 for control and V-BrPO treated lignin phenols, respectively. ........................................................................... 170
Figure 7-2. Fluorescence of V-BrPO treated CDOM collected from (a) Achnanthes longipes (orange), (b) Skeletonema costatum (purple), (c) Synechococcus elongates (green), and (d) Synechococcus sp. (pink) as a function of time. Fluorescence of their controls is presented in grey symbols. Error bars indicate 1 standard deviation (± 1σ) of replicates, number of samples (n) were 3 and 2 for control and V-BrPO treated CDOM collected from phytoplankton cultures, respectively. Note the different time scales of S. costatum and Synechococcus sp. .................................................................... 171
Figure 8-1. A conceptual model of BrVSLS production from within the phycosphere to the surrounding environment. ............................................... 179
Figure 8-2. CHBr3 concentrations observed from halogenated artificial seawater (HASW) buffered to different pHs. ................................................................ 181
xv
LIST OF TABLES
Page Table 2-1. Global annual averaged atmospheric lifetimes for CHBr3, CH2Br2, and
CHClBr2 (WMO [2003]and references therein). ................................................. 8
Table 2-2. R2 (p < 0.01) for correlations between CHBr3, CH2Br2, and CHClBr2 seawater concentrations and atmospheric mixing ratios during the GOMECC cruise. ............................................................................................. 14
Table 2-3. Air mixing ratios, surface water concentrations, saturation anomalies, fluxes and calculated production rates for CHBr3, CH2Br2, and CHClBr2 for the GOMECC cruise. .................................................................................. 18
Table 2-4. CH2Br2/CHBr3 air and seawater concentration ratios. ................................... 22
Table 2-5. A comparison of the VSLS fluxes (nmol m-2 d-1) from this study with Sweeney et al. [2007] (S07), Nightingale et al. [2000] (N00) and Wanninkhof [1992] (W92) parameterizations and previous studies. ................ 27
Table 3-1. Mean (range; number of samples) BrVSLS concentrations (pmol L-1) in coastal and open ocean water column. Bold text highlighted the minimum and maximum of the data. ................................................................................ 47
Table 3-2. Spearman’s rank correlation coefficient (ρ) of BrVSLS with picoplankton, p-value and number of samples (n) are presented in the parentheses. Relationships between the BrVSLS with photosynethic picoplanktons were not considered below the euphotic zone. Below the euphotic zone, only relationships between BrVSLS and heterotrophic bacteria were considered. “ns” indicates not significant correlation. ............... 56
Table 3-3. Spearman’s rank correlation coefficient (ρ) of CHBr3 with CH2Br2, CHClBr2, and CHBrCl2, p-value and number of samples (n) are presented in the parentheses. “ns” indicates not significant correlation. .......................... 60
Table 4-1. List of cruises presented in this study. ............................................................ 67
Table 4-2. Mean (range) of BrVSLS atmospheric mixing ratios, seawater concentrations, saturation anomalies, and fluxes during the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 in different hemispheres and seasons. ............................................................................................................. 77
Table 4-3. Mean (ranges) of BrVSLS atmospheric mixing ratios, seawater concentrations, saturation anomalies, and fluxes during the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises in different environmental regimes. Open ocean is defined as water depth > 200 m, mid-ocean island
xvi
is defined as 200 km radius of mid-ocean islands, and coastal ocean is defined as water depth ≤ 200 m. ....................................................................... 87
Table 4-4. Extrapolated global open ocean net sea-to-air fluxes of CHBr3 and CH2Br2 (Gmol Br yr-1), based on data from the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises. ................................................................. 88
Table 4-5. Spearman’s rank correlation coefficient (ρ) between chlorophyll a concentrations with CHBr3 and CH2Br2 open ocean seawater concentrations, based on data from the A16N, A16S, HalocAST-A, BLAST-II, and GasEx98 cruises, and all data combined. p-value and number of samples (n) are in parentheses. “ns” indicates the correlation is not significant. Note that unless it is indicated in the table, correlation coefficient is based on satellite observed chlorophyll a data. .......................... 96
Table 4-6. Spearman’s rank correlation coefficient (ρ) between biological variables with CHBr3 and CH2Br2 open ocean seawater concentrations during the HalocAST-A cruise, p-value and number of samples (n) are in parentheses. “ns” indicates the correlation is not significant. Only parameters exhibit significant correlation (p < 0.05) are shown. ..................... 99
Table 5-1. List of model DOM compounds used in this study with their chemical formula, with final DOC concentrations (µM) after added to artificial seawater (ASW), and summary of BrVSLS production properties. “N/A” indicates bulk macromolecule without a defined chemical formula. “Interfered” notes DOM that yielded significantly less BrVSLS than V-BrPO treated ASW. “No observable effect” notes addition of DOM did not enhance or interfere with BrVSLS production, relative to V-BrPO treated ASW. “Enhanced” notes DOM that is capable of producing significantly more BrVSLS than V-BrPO treated ASW. It should be note that DOM that interfered with BrVSLS production is not due to inhibition of V-BrPO. ..................................................................................................... 110
Table 5-2. BrVSLS concentrations measured after 4-hours reaction of V-BrPO treated artificial seawater (ASW), aged Gulf of Mexico seawater, and samples with model DOM compounds added to ASW. ................................. 117
Table 5-3. Spearman rank correlation coefficient (ρ) between the BrVSLS (p-value; number of samples n). .................................................................................... 120
Table 6-1. Phytoplankton species screened for BrVSLS and CHCl3 production. “+” indicates significant production of BrVSLS and CHCl3 were observed in the culture. “-” indicates there were no observable production of BrVSLS and CHCl3 in the culture. “c.d.” notes production cannot be determined due to co-elution of unidentified substances with the compounds of interested. ........................................................................................................ 145
xvii
Table 6-2. Concentrations (±1 standard deviation) of CHBr3, CH2Br2, CHClBr2, CHBrCl2, and CHCl3 produced in A. longipes and Synechococcus sp. cultures. .......................................................................................................... 153
1
CHAPTER I
INTRODUCTION
Bromoform (CHBr3), dibromomethane (CH2Br2), chlorodibromomethane
(CHClBr2), and bromodichloromethane (CHBrCl2) comprise the natural brominated
very short-lived substances (BrVSLS). CHBr3 and CH2Br2 account for ~80% of the
very short-lived organic bromine in the marine boundary layer [Law and Sturges,
2007]. BrVSLS degrade into inorganic bromine (Bry), which can catalytically destroy
ozone, via photolysis and reaction with hydroxyl radicals (OH) in the atmosphere.
These compounds are receiving increased interest, as the BrVSLS can supply
significant amounts of Bry to the stratosphere, in addition to the relatively longer-lived
ozone depleting substances (ODSs), such as methyl bromide (CH3Br) and halons. In
addition, bromine is ~50 to 100% more effective in destroying ozone than chlorine, on
an atom-by-atom basis [Garcia and Solomon, 1994; Solomon et al., 1995].
Bromine atoms can enter the stratosphere via either source gas injection (SGI)
or product gas injection (PGI). SGI is defined as the trace gases entering the
stratosphere in their original forms as they were emitted. In regions experiencing
strong atmospheric convection, such as the intertropical convergence zone (ITCZ),
short-lived gases can be rapidly delivered to the stratosphere as source gases [Montzka
and Reimann, 2011]. PGI on the other hand, is defined as delivering degradation
products of trace gases to the stratosphere. Overall, relative contributions from SGI
and PGI are mainly determined by the atmospheric lifetimes of the various trace gases
and their product gases, and atmospheric transport [Montzka and Reimann, 2011].
2
Although BrVSLS tend to be degraded significantly in the troposphere, their
degradation products are even less stable in the atmosphere allowing for substantial
further degradation (e.g. chemical and physical scavenging) [Hossaini et al., 2012b].
Hence, product gases of CHBr3 and CH2Br2 may be removed rapidly from the
atmosphere before they can reach the stratosphere via PGI. Therefore, SGI is thought
to be the major delivery mechanism for Br CHBr3 and CH2Br2 to the stratosphere
[Hossaini et al., 2012b].
These natural BrVSLS can contribute ~1 to 8 parts per trillion (ppt) of Bry to
the stratosphere, which is equivalent to ~4 to 36% of total stratospheric bromine,
which included organic and inorganic bromine in the stratosphere [Montzka and
Reimann, 2011]. This portion of Bry is also known as VSLS supplied Bry (BryVSLS),
where CHBr3 and CH2Br2 together account for 76% of BryVSLS [Hossaini et al.,
2012a]. As production of the anthropogenic ozone depleting substances (ODSs) has
been phased out under the Montreal Protocol and their concentrations are continuing
to decrease in the atmosphere, the naturally produced ODSs are becoming more
important. As anthropogenic ODS concentrations decline, the relative contribution of
BrVSLS to stratospheric bromine will increase. Ultimately, natural BrVSLS will
become the key in controlling stratospheric ozone chemistry.
In a future climate scenario, the predicted stronger atmospheric convection is
likely to enhance transport of CHBr3 to the stratosphere as source gas injection (SGI)
[Hossaini et al., 2012b]. SGI for CH2Br2, CHClBr2, and CHBrCl2 is also likely to
increase due to possible reduction in OH concentration, resulting from the predicted
increase in atmospheric methane (CH4) concentration [Hossaini et al., 2012b].
3
Therefore, knowing where these natural BrVSLS are being produced and their
emission strengths are essential for understanding ozone chemistry now and in the
future. A fundamental question exists: how (why) are natural BrVSLS produced?
While enzyme-mediated halogenation by vanadium bromoperoxidase (V-
BrPO) is a widely accepted mechanism for BrVSLS production, it has been largely
neglected by the trace gas community. Little information is available on the factors
controlling the production of BrVSLS. BrVSLS production is frequently related to
photosynthetic biomass, such as macroalgae and phytoplankton [Carpenter and Liss,
2000; Goodwin et al., 1997; Manley et al., 1992; Moore et al., 1996; Sturges et al.,
1992; Sturges et al., 1993]. To produce CHBr3, CHClBr2, CHBrCl2, and supposedly
CH2Br2, three major components are required: (1) photosynthetic biomass that is
capable of producing V-BrPO; (2) hydrogen peroxide (H2O2) to initiate the formation
of hypobromous acid (HOBr); (3) suitable organic matter (OM) to be halogenated by
HOBr. The V-BrPO induced HOBr (HOBrenz) in the presence of H2O2 can halogenate
a wide range of OM and form halogenated organic molecules. Finally, some of the
unstable halogenated organic molecules can release BrVSLS via carbon-carbon bond
cleavage in an alkaline environment, such as in seawater (pH ~8.1) [Butler and
Carter-Franklin, 2004; Theiler et al., 1978].
4
For a decade, researchers have been focusing on “who” are the BrVSLS
producers [Abrahamsson et al., 2004; Carpenter and Liss, 2000; Colomb et al., 2008;
Liu et al., 2013; Liu et al., Submitted; Mattson et al., 2012; Quack et al., 2007a]. Only
a few studies investigated the impact of H2O2 concentration and dissolved organic
matter (DOM) on BrVSLS production [Abrahamsson et al., 2003; Lin and Manley,
012; Manley and Barbero, 2001]. This dissertation focuses on two major aspects of
BrVSLS biogeochemistry in the ocean: (1) describing how the BrVSLS are distributed
in the global ocean, at the surface and in the water column (Chapters II, III, and IV);
(2) determining what drives the observed distribution and variability (Chapters V and
VI). Finally, I also present the significant implications of V-BrPO halogenation of
DOM for the marine carbon cycle (Chapter VII). This research not only provides
insight to marine source of BrVSLS, but also elucidates brominated organic matter
biogeochemistry in seawater (Figure 1-1).
5
Figure 1-1. Brominated organic matter in marine and atmospheric environments derived from a conceptual model of V-BrPO enzyme-mediated BrVSLS formation mechanisms. Marine algae that produce V-BrPO can oxidize bromine to form reactive species such as hypobromous acid (HOBr), in the presence of H2O2. HOBr can react with a wide variety of DOM compounds and consequently form brominated DOM. Some brominated DOM may release BrVSLS and contribute to catalytic ozone destruction reactions; but some brominated DOM maybe stable enough and can potentially be sorbed to sinking particulates; hence being transported into deeper waters. Some of the brominated OM on the particulates may be stable enough to resist degradations in the water column and in the sediments. Atmospheric interactions shown in this figure is adapted from Salawitch [2006].
6
CHAPTER II
CHBr3, CH2Br2 AND CHClBr2 IN U.S. COASTAL WATERS DURING THE
GULF OF MEXICO AND EAST COAST CARBON (GOMECC) CRUISE*
2.1 Introduction
Brominated very short live substances (VSLSs) such as bromoform (CHBr3),
dibromomethane (CH2Br2), chlorodibromomethane (CHClBr2), etc. are important
bromine carriers to the stratosphere in addition to the relatively more stable
compounds such as halons and methyl bromide (CH3Br) [Kurylo and Rodriguez,
1999]. These VSLSs degrade and release inorganic bromine (Bry) via photolysis and
reaction with hydroxyl radicals (OH) [WMO, 2007]. The resulting Bry can participate
in catalytic ozone destruction in the free troposphere [von Glasow et al., 2004; Yang et
al., 2005] and the stratosphere (e.g. Garcia and Solomon [1994], McElroy et al.
[1986], and Solomon et al. [1995]). Bromine is 40 to 100 times more efficient in O3
destruction than chlorine (Wamsley et al. [1998] and references therein). The VSLSs
are capable of supplying ~5.0 – 5.2 pptv of Bry to the stratosphere [Dorf et al., 2008;
Sinnhuber et al., 2002]. CHBr3 and CH2Br2 are the most abundant VSLSs, which,
when combined, account for > 80% of VSL organic bromine in the marine boundary
layer and the upper troposphere [WMO, 2007]. CHBr3 alone could contribute ~0.5 to
* Reprinted with permission from “CHBr3, CH2Br2, and CHClBr2 in U.S. coastal waters during the Gulf of Mexico and East Coast Carbon cruise” by Y. Liu et al., 2011. Journal of Geophysical Research, Oceans, 116, C10004, doi:10.1029/2010JC006729., Copyright [2011] by John Wiley and Sons.
7
1.8 pptv of Bry to the stratosphere [Dvortsov et al., 1999; Nielsen and Douglass, 2001;
Sinnhuber and Folkins, 2006].
Current evidence suggests that marine CHBr3, CH2Br2, and CHClBr2 are
primarily produced naturally, such as by macroalgae, ice algae and phytoplankton (e.g.
Carpenter and Liss [2000]; Gschwend and MacFarlane [1986]; Manley and Barbero
[2001]; Manley et al. [1992]; Moore et al. [1996]; Sturges et al. [1992]; Sturges et al.
[1993]; and Tokarczyk and Moore [1994] etc.). CHBr3 and CHClBr2 also have
anthropogenic sources as by-products of water disinfection (e.g. Nieuwenhuijsen et al.
[2000]; Quack and Wallace [2003]; Ram et al. [1990]). Sea-to-air flux is the major
source of atmospheric CHBr3 in the marine environment [Carpenter and Liss, 2000;
Quack and Wallace, 2003]. However, Carpenter et al. [2007b] also indicated sea-to-
air flux of CHBr3 alone could not account for the high atmospheric mixing ratios
observed in the tropical Atlantic. The authors suggested non-localized sources,
possibly terrestrial contributions for CHBr3.
The temporal and spatial distributions of these compounds vary significantly,
especially for CHBr3, which has the shortest overall atmospheric lifetime (Table 2-1)
(WMO [2003] and references therein). These variations are attributable to
geographical distributions of source types, source strengths, gas-exchange coefficients,
local degradation rates, and seasonality (e.g. Carpenter et al. [2009]; Hughes et al.
[2009]; Liang et al. [2010]; Quack and Wallace [2003]; Warwick et al. [2006]; and
WMO [2007] etc.). Such variations and the limited availability of paired seawater and
atmospheric measurements of the VSLSs lead to large uncertainties for estimating
their global fluxes. Attempts to estimate global coastal fluxes have even larger
8
uncertainties due to even more variability in the coastal environments. Coastal areas
are important elements for VSLSs global fluxes. Quack and Wallace [2003] reported a
CHBr3 global coastal (shore regime) flux that accounts for ~23% of their estimated
global flux. Liang et al. [2010] reported model stimulated CHBr3 and CH2Br2 global
fluxes of which 40% is from the coasts. Carpenter et al. [2009] also reported that their
highest CHBr3 and CH2Br2 fluxes were observed in the coastal waters in the northeast
Atlantic, and tropical eastern Atlantic. In this study, we present the first CHBr3,
CH2Br2, and CHClBr2 data over an extensive area of the coastal United States (24º –
43º N). This is the first brominated VSLSs data set collected from large area of
urbanized coastline.
Table 2-1. Global annual averaged atmospheric lifetimes for CHBr3, CH2Br2, and CHClBr2 (WMO [2003] and references therein).
τOH
(day) τJ
(day) τtotal
(day) CH2Br2 120 5000 120 CHClBr2 120 161 69 CHBr3 100 36 26
2.2 Methods
For the Gulf of Mexico and East Coast Carbon (GOMECC) cruise, the NOAA
ship, R/V Ronald H. Brown, departed from Galveston, TX on July 10 and arrived in
Boston, MA on August 4, 2007 (Figure 2-1). Surface air and water samples were
collected and analyzed for CHBr3, CH2Br2, CHClBr2, and a suite of other halocarbons.
9
Continuous air and surface water samples were collected from Year Day (YD) ~196
onward.
Figure 2-1. Map showing the Gulf of Mexico and East Coast Carbon (GOMECC) cruise with track (▬), 200m isobath (▬), yearday (●), treated water outfalls (♦), and seawater-cooled nuclear power plants (♦).
Air was continuously pumped (~6 L min-1) through a 0.63-cm ID SynFlex
tubing (Motion Industries, Dallas, TX), which was mounted on the mast at the bow of
the ship. Seawater from the ship’s pumping system flowed through a Weiss
equilibrator [Butler et al., 1988; Johnson, 1999]. Samples are periodically drawn from
the equilibrated headspace of the Weiss equilibrator and from the air pumped from the
10
bow. Air and equilibrator headspace samples were collected alternately every ~56, or
112 minutes if the calibration gas standard is in sequence. Each sample was drawn
through a Nafion dryer, and preconcentrated on one cryotrap and focused on a second
cryotrap. The sample is then injected into a ~60 m, 0.25mm DB – VRX (J & W)
column and analyzed by Gas Chromatography and Mass Spectrometry (GC-MS)
[Yvon-Lewis et al., 2004]. The equilibrium partial pressure (pw) of each gas is
corrected for any warming effects induced by the ship’s pumping system. Calibration
standards, run every fifth injection, are whole air working standards calibrated against
gravimetric standards prepared at NOAA Earth System Research Laboratory (ESRL).
For CHClBr2, the working standards were calibrated against liquid standards dissolved
in ~35 salinity synthetic seawater in the lab. The analytical system performance was
determined by working standard analysis results. The detection limits for CHBr3,
CH2Br2, and CHClBr2 were 0.02 ppt, 0.04 ppt, and 0.01 ppt respectively. The total
measurement uncertainties for CHBr3, CH2Br2, and CHClBr2 were 7.31%, 6.34% and
8.79% respectively.
Chlorophyll-a (chl-a) and colored dissolved organic matter (CDOM) were
measured with a Wetlab “ECO” triplet sensor, which was calibrated in the
manufacture before the GOMECC cruise. The instrument with factory calibration
settings was submerged in an 80 L black tank supplied with seawater from the ship’s
pumping system at a rate of 4 to 7 L min-1. The sensors measured the stimulated
fluorescence of chlorophyll-a (µg L-1, optical configuration: ex470/em685) and
CDOM (quinine sulfate units, optical configuration: ex370/em460) at a rate of 1 Hz,
and the signal was averaged over 1 minute.
11
2.3 Results and discussions
2.3.1 Oceanic and atmospheric conditions
Our data cover the eastern Gulf of Mexico and the western Atlantic coastlines
of the United States of America. Much of the cruise track is located in water shallower
than 200 m (Figure 2-1). Three major oceanic currents in the study region are the
Loop Current in the Gulf of Mexico, the northward Gulf Stream as a continuation of
the Loop Current, and the Labrador Current moving south along the coast at higher
latitudes. Most of the data collected in the Gulf of Mexico are under the influence of
coastal seawater, while some of the offshore data were collected in the Loop Current.
Most of the data from the Florida Straits to just south of Cape Hatteras (Lat ~ 35.7ºN)
could experience influence from the Gulf Stream. Abrupt changes in sea surface
temperature (SST) and salinity were observed around YD 207 (Lat ~ 36.8ºN) (Figure
2-2a). This distinct feature indicated the influence of the Labrador Current, which
brought colder and fresher water southward along the coastline.
12
Figure 2-2. Time series of salinity (▬) and sea-surface temperature (▬) (a), chlorophyll a concentrations (▬) (b), CDOM concentrations (▬) (c), and wind speed (▬) (d) for the GOMECC cruise. The gray shadings highlight open ocean data.
In the atmosphere, the subtropical high pressure over the Atlantic basin was
governing the general atmospheric circulation in our study area. This atmospheric
feature led to mostly onshore winds along the eastern Florida coast, and alongshore
winds north of Cape Canaveral, Florida until YD 202. On YD 202 and onward, the
13
coastal atmospheric circulation patterns were more variable with occasional offshore
winds, which could bring continental air masses into the study region (Figure 2-3).
Figure 2-3. Daily 48 hr 100m above sea surface Hysplit air mass back trajectories for selected points (▬) along the GOMECC cruise track (▬), and back trajectories for selected points with negative saturation anomalies (YD 199 – 200) (▬). (★) indicated the starting point of the back trajectories.
14
2.3.2 Atmospheric mixing ratios, seawater concentrations and saturation
anomalies
The continuously alternating air and equilibrator headspace sampling allows us
to examine the relationship between the VSLS water concentrations and the mixing
ratios in the adjacent air. The mean atmospheric mixing ratios for CHBr3, CH2Br2, and
CHClBr2 were 14.6 (0.7 to 138.3) ppt, 2.8 (0.5 to 13.2) ppt, and 0.5 (0.03 to 3.2) ppt,
respectively. The mean water concentrations for the entire GOMECC cruise were 66.0
(4.4 – 1724.8) pmol L-1, 10.6 (1.9 – 153.8) pmol L-1, and 1.0 (0.1 – 17.2) pmol L-1, for
CHBr3, CH2Br2 and CHClBr2, respectively. The VSLSs have strong correlations with
each other in the seawater for the entire cruise (Table 2-2). This suggests they may
share common sources, which is consistent with previous studies (e.g. Carpenter
[2003]; Laturnus [1996]; and Yokouchi et al. [2005]). However, these VSLSs are less
correlated with each other in the atmosphere than in seawater (Table 2-2). This may be
due to atmospheric mixing and differences in atmospheric lifetimes (Table 2-1).
Table 2-2. R2 (p < 0.01) for correlations between CHBr3, CH2Br2, and CHClBr2 seawater concentrations and atmospheric mixing ratios during the GOMECC cruise.
Seawater Air R2 CH2Br2 CHClBr2
CH2Br2 CHClBr2
CHBr3 0.93 (n = 280) 0.95 (n = 276)
0.78 (n = 279) 0.63 (n = 275) CH2Br2 - 0.96 (n = 275) - 0.37 ( n = 274)
15
Atmospheric mixing ratios and surface water concentrations for CHBr3,
CH2Br2, and CHClBr2 generally exhibit similar trends (Figures 2-4a and b), except the
data collected during YD ~199 to 200. During this period, atmospheric mixing ratios
were significantly elevated (up to 56.6 ppt for CHBr3), with no corresponding
elevation in the water concentrations resulting in negative saturation anomalies in
some locations along the transit (Figure 2-5a). The Saturation anomaly (SA; %) of a
gas indicates the deviation from equilibrium between the ocean and the atmosphere,
and is calculated with the following equation:
SA = Pw -‐ PaPa
×100% (Equation. 2.1)
where pw (atm) is the equilibrium partial pressure of the gas dissolved in surface
seawater, and pa (atm) is the partial pressure of the gas in the air.
The 48 hour 100 m above sea surface HYSPLIT (http://www.arl.noaa.gov/
HYSPLIT.php) back trajectories for YD 199 to 200 indicate that the high atmospheric
mixing ratios near the Florida Straits and the east coast of Florida that led to negative
saturation anomalies may be the result of air mass transport from the southeast of the
ship, whereas the back trajectories for locations before and after the elevated
atmospheric mixing ratios (YD 198 and 201) indicate different air mass source regions
(Figure 2-3). The seawater was supersaturated and served as a source of the VSLSs to
the atmosphere everywhere else during the GOMECC cruise.
16
2.3.3 Coastal sources and sinks
Data that were collected when the water depth was less than or equal to 200
meters are extracted and are considered as coastal data. This is similar to the
combined coastal and coastally influenced region defined by Carpenter et al. [2009]
and the shelf region defined by Quack and Wallace [2003]. The mean coastal water
concentrations are higher than the averages for the open ocean concentrations (Table
2-3). These findings suggest there are significant sources of VSLSs to the coastal
ocean, which agrees with earlier findings for CHBr3 and CH2Br2 [Carpenter et al.,
2009; Quack and Wallace, 2003].
2.3.3.1 Anthropogenic sources
The Gulf of Mexico and east coast United States of America are highly
urbanized regions. Human inputs could influence the VSLS budgets in various ways,
such as treated water outfalls and seawater-cooled nuclear or conventional power
plants. These facilities discharge significant amounts of chlorinated (for disinfection)
fresh water or seawater; and it is well known that the chlorine disinfection processes in
either fresh water or seawater could produce a wide range of byproducts, such as the
trihalomethanes (e.g. Nieuwenhuijsen et al. [2000]; Quack and Wallace [2003]; Ram
et al. [1990]). Massive nutrient inputs from the outfalls could stimulate microalgal or
macroalgal blooms [Lapointe et al., 2004; Lapointe et al., 2005], which may, in turn,
produce the VSLSs.
17
During the GOMECC cruise, we encountered two nuclear power plant cooling
water discharge areas and a series of treated water outfalls along the east coast of
Florida, and the treated water outfall in Boston Harbor. Elevated VSLS concentrations
were observed in the seawater when the ship passed these locations. Two spikes in
surface water concentrations (Figure 2-4b), one just before YD 200 and one after that
day coincide with the time when the ship passed near a series of nuclear power plants
and treated-water outfalls along the Florida coast. The highest water concentrations for
the VSLSs were seen near the Boston Harbor (on YD ~210). These findings suggest
anthropogenic inputs. However, it is impossible to estimate the magnitude of the
contribution from these facilities for several reasons. First, we do not have tracers that
allow us to identify waters discharged from these facilities. Nutrient data might have
been a good tracer for treated water outfalls plumes. Unfortunately, these data are not
available for the locations of interest during the GOMECC cruise. Therefore, it would
be difficult for us to estimate the extent of treated water outfall influence to the water
mass that we sampled. The features that we observed in these areas are likely to be
mixtures of anthropogenic sources, and co-existing natural sources as describe below.
18
Table 2-3. Air mixing ratios, surface water concentrations, saturation anomalies, fluxes and calculated production rates for CHBr3, CH2Br2, and CHClBr2 for the GOMECC cruise.
Air Mixing Ratio (ppt)
Surface Water Conc. (pmol L-1)
Saturation Anomaly (%)
Calculated Production Rates (nmol m-3 d-1)
Open Ocean
CHBr3 14.9
(0.8 - 47.4) 39.7
(4.4 - 125.2) 282.5
(-14.7 - 1201.8) 2.4 ± 1.1*
(-1.0 - 22.5)
CH2Br2 2.7
(0.5 - 9.2) 6.6
(1.9 - 17.7) 301.5
(-12.0 - 775.5) 3.4 ± 1.1*
(1.1 - 12.5)
CHClBr2 0.5
(0.04 - 3.2) 0.7
(0.1 - 2.1) 252.0
(-48.1 - 1129.0) 0.03 ± 0.003*
(-0.1 - 0.6)
Coastal (≤ 200m)
South of 40°N
CHBr3
15.4 (0.9 - 138.3)
41.0 (6.0 - 366.8)
281.9 (-27.7 - 1274.7)
6.1 ± 1.1* (-2.6 - 36.4)
CH2Br2 2.8
(0.6 - 13.2) 7.5
(2.3 - 37.3) 326.1
(25.6 - 1317.4) 4.8 ± 1.1*
(1.4 - 21.8)
CHClBr2 0.5
(0.05 - 2.6) 0.8
(0.2 - 3.9) 303.1
(-25.0 - 1141.6) 0.1 ± 0.003* (-0.04 - 0.8)
North of 40°N
CHBr3
12.5 (0.7 - 90.7)
162.5 (4.7 - 1724.8)
390.4 (76.5 - 1536.3)
31.2 ± 1.1* (0.4 - 395.0)
CH2Br2 3.0
(0.5 - 10.8) 23.9
(1.9 - 153.8) 428.4
(61.7 - 2002.1) 14.2 ± 1.1*
(1.1 - 101.7)
CHClBr2 0.3
(0.03 - 1.3) 2.1
(0.1 - 17.2) 505.6
(-83.3 - 2329.7) 0.3 ± 0.003* (-0.1 - 3.6)
* Uncertainties for calculated production rates for CHBr3, CH2Br2, and CHClBr2 calculated using error propagating method with uncertainties given for each degradation rate constant.
19
Figure 2-4. Time series of atmospheric mixing ratios (a) and water concentrations (b) for CHBr3 (□), CH2Br2 (+), and CHClBr2 (×). The gray shadings highlight open ocean data.
2.3.3.2 Natural sources
Several studies reported that seawater CHBr3 concentrations are generally
positively correlated with chlorophyll a (Carpenter et al. [2009]; and Schall et al.
[1997] etc.). Manley and Barbero [2001] suggested the presence of certain types of
dissolved organic matter (DOM) may result in CHBr3 production. However, our
results found that the VSLS concentrations over the whole study region show no clear
relationships with chlorophyll a (R2 < 0.1) or colored dissolved organic matter
(CDOM) (R2 < 0.05) (Figures 2-2 b and c).
Low correlation between CHBr3 concentrations in seawater and CDOM may
be attributable to the complicated coastal DOM components, as the type of DOM
20
required for trihalomethane formations may be very specific. For example, Theiler et
al. [1978] suggested ketones are better substrates for CHBr3 formations than phenols.
The low correlation between CHBr3 concentrations in seawater and chlorophyll a is in
agreement with Hughes et al. [2009], in which observations were made in coastal
Antarctica during the summer. However, the R2 values reported by the referenced
study for CHBr3 and CH2Br2 (R2 < 0.4) with chlorophyll a were still much higher than
those observed in this study. Chlorophyll a analysis is not species specific or able to
detect the presence of macroalgae in the surrounding area. Hence, such a relationship
could be altered by co-existing non-bromoalkane producing species or differences in
bromoalkane productivities between each species that were present in the water.
Hughes et al. [2009] also suggested the effects of sea-to-air gas exchange could alter
the correlation. Other explanations for the lack of correlation could also be
anthropogenic inputs and potential macroalgal contributions in some areas.
The area north of 40ºN in our study region is known to support numerous
macroalgae species [Lüning, 1990]. Elevated VSLS concentrations in this area may be
attributed to macroalgal production, in addition to the anthropogenic sources discussed
above. Macroalgal influence south of this area is uncertain. Rocky bottoms that are
favorable for macroalgae growth are almost entirely absent from south of Long Island
until the Florida Keys (Figure 2-1), where the lime stone bottom could provide
anchorage for the rootless macroalgae [Searles, 1984]. Between these areas,
macroalgae growth is dependent on outcrops and near shore human-made structures,
which could restrict macroalgae growth in places very close to the coastline in a
21
patchy pattern. Therefore, during the transit between 30ºN to 40ºN, we may not have
been close enough to the macroalgal habitats.
2.3.3.3 CH2Br2/CHBr3 ratios
In previous studies, CH2Br2/CHBr3 concentration ratios in seawater and in air
were lower in coastal regions than in open ocean regions [Carpenter et al., 2009;
Quack et al., 2007b]. The drivers of these ratios could be related to the first order
decay of CHBr3 and CH2Br2 in the atmosphere and seawater, and emission ratios
which themselves differ from source to source [Carpenter et al., 2009]. However, we
did not see significant differences between the ratios of CH2Br2/CHBr3 in the coastal
waters and the open ocean for either seawater concentrations or atmospheric mixing
ratios (Table 2-4). This may be due to the fact that we have limited data to represent
open ocean characteristics, and hence, could not make legitimate comparisons with
other studies in the open ocean. The wide ranges of air and seawater concentration
ratios shown in Table 2-4 suggest there are substantial heterogeneities in the sources
and sinks in this extensive study area. The highest water and air concentration ratios
observed during this cruise are in the coastal region north of 40ºN, which is likely to
have the most macroalgal influence. This is contrary to the results from earlier studies
suggesting lower ratios in areas of macroalgal influence [Carpenter et al., 2009].
22
Table 2-4. CH2Br2/CHBr3 air and seawater concentration ratios.
Water Air
Open Ocean 0.2 (0.1 - 0.4)
0.3 (0.05 - 0.7)
Coastal South of 40°N 0.2
(0.06 - 0.4) 0.3
(0.07 - 0.7)
North of 40°N 0.3 (0.08 - 0.7)
0.4 (0.1 - 0.8)
2.3.3.4 CHBr3 photodegradation in surface seawater
Different degradation rates in seawater may also contribute to the different
CH2Br2/CHBr3 concentration ratios observed in our study. Photolysis of CHBr3 in the
mixed layer could lead to a higher CH2Br2/CHBr3 ratio in water concentrations in the
area north of 40°N.
Carpenter and Liss [2000] and Hense and Quack [2009] briefly discussed the
role of photolytic loss of CHBr3 in surface seawater. Carpenter and Liss [2000]
estimated the mixed layer CHBr3 1/e lifetime due to photolysis is ~9 years in a 100 m
mixed layer, assuming that all of the photochemical reactions occur only in the top 1
m, and assuming a photolysis lifetime of ~30 days in the top layer. Their findings
suggested that CHBr3 photolysis loss is negligible in situations with deep mixed
layers, such as in the open ocean.
However, photolysis of CHBr3 may be a significant sink in this study region.
For the GOMECC cruise, the mean mixed layer depth in the region north of 40ºN is
only ~3.6 (2.0 – 7.0) m, while the mean mixed layer depth in the region south of 40ºN
is ~ 13.2 (2.0 to 50.0) m. Therefore, the photodegradation rate constant for CHBr3 (Jz)
23
is calculated to assess the significance of the photolytic loss of CHBr3 to its overall
budget in coastal waters during the GOMECC cruise.
The modeled lifetime for the top 1 m layer is ~ 25 days, which is relatively
close to the referenced study [Carpenter and Liss, 2000]. However, the COART [Jin
et al., 2004] model used for determining percentage of light attenuation at each layer
relative to the atmosphere indicated that the wavelengths that are capable of
photolytically degrading CHBr3 can penetrate down to 5 m for conditions experienced
during the GOMECC cruise. The CHBr3 mean integrated lifetime for the top 5 m of
the mixed layer is ~ 34 days. Applying such rate constant to the open ocean and
assuming a mixed layer depth of 100 m, the total mixed layer lifetime is shortened to ~
2 years.
The mean mixed layer depth during this cruise is almost ten times shallower
than the typical open ocean mixed layer depth. The mean mixed layer photolysis
lifetime calculated for the GOMECC data is ~82 days, which is much shorter than the
lifetime in the open ocean. Since 34% of our data were collected in locations with
mixed layer depths less than 5 m, where the whole mixed layer experienced
photodegration of the compound, photolytic loss is an important component of the
budget for seawater CHBr3 during the GOMECC cruise. Although this modeled
photolysis loss in seawater is rather crude, it indicates the importance of this
degradation path in coastal environments.
24
2.3.4 Net fluxes
The net sea-to-air fluxes (F, nmol m-2 d-1) for the bromoalkanes are calculated
with the following equation:
F = 𝑘𝑘w(Cw − !a!) (Equation 2.2)
where kw (m d-1) is the wind speed dependent gas exchange coefficient Sweeney et al.
[2007] corrected for the Schmidt number of the gas of interest; H (m3 atm mol-1) is the
Henry’s law constant [Moore et al., 1995a]; Cw (mol m-3) is the concentration of the
gas of interest in the surface seawater; and pa (atm) is the partial pressure in the air.
Wind speeds were measured at ~19 m and were corrected to 10m above the sea
surface [Erickson, 1993] for the flux calculations. The Schmidt numbers for the
VSLSs were calculated from the kinematic viscosity of seawater and diffusivities of
the gases [Hayduk and Laudie, 1974].
The sea-to-air fluxes of CHBr3, CH2Br2 and CHClBr2 reflect the effects of
wind speeds (Figure 2-2d) and concentration gradients between water and air. For the
entire cruise, the mean fluxes for CHBr3, CH2Br2, and CHClBr2 were 47.6 (-25.4 to
1056.3) nmol m-2 d-1, 9.7 (-0.5 to 112.3) nmol m-2 d-1, and 0.8 (-1.2 to 10.8) nmol m-2
d-1, respectively, assuming the Sweeney et al. [2007] parameterization for the gas
exchange coefficient as our best estimate (Figure 2-5b). The coastal mean net fluxes
for these bromoalkanes were significantly higher than the open ocean net fluxes
(Table 2-5). The overall coastal net fluxes for CHBr3, CH2Br2, and CHClBr2 were 59.0
(-25.4 to 1056.3) nmol m-2 d-1, 11.5 (0.3 to 112.3) nmol m-2 d-1, and 1.0 (-0.4 to 10.8)
nmol m-2 d-1, respectively.
25
Figure 2-5. Time series of saturation anomalies (a), fluxes (b), and calculated production (c) for CHBr3 (□), CH2Br2 (+), and CHClBr2 (×). The gray shadings highlight open ocean data.
As discussed above, we consider the area north of 40º N as predominantly
influenced by macroalgal and anthropogenic inputs. The maxima for the sea-to-air
fluxes of the gases were observed in this region. The mean coastal net fluxes in the
area north of 40ºN were 85.1 (0.6 to 1056.3) nmol m-2 d-1 for CHBr3, 11.2 (0.3 –
112.3) nmol m-2 d-1 for CH2Br2, and 1.0 (-0.3 to 10.8) nmol m-2 d-1 for CHClBr2. The
mean sea-to-air fluxes for CHBr3 and CH2Br2 in this area were significantly lower
26
than the mean fluxes reported by Jones et al. [2009] and Zhou et al. ([2005] and
[2008]) (Table 2-5), where measurements were made in locations that may have been
influenced by macroalgal production. The area south of 40º N is not thought to be
macroalgae dominated, and it is more likely to be influenced by phytoplankton and
anthropogenic sources. The coastal net fluxes for the VSLSs in this region were 45.7
(-25.4 to 334.3) nmol m-2 d-1, 11.6 (1.0 to 62.0) nmol m-2 d-1, and 1.0 (-0.4 to 5.4)
nmol m-2 d-1, for CHBr3, CH2Br2, and CHClBr2, respectively. CHBr3 and CH2Br2
coastal net fluxes in this region are approximately half of those reported by Hughes et
al. [2009] during the Antarctic summer at locations dominated by ice algae. These
fluxes are also significantly lower than fluxes observed by Carpenter et al. [2009]
along the tropical Atlantic coast (Table 2-5).
27
Table 2-5. A comparison of the VSLS fluxes (nmol m-2 d-1) from this study with Sweeney et al. [2007] (S07), Nightingale et al. [2000] (N00) and Wanninkhof [1992] (W92) parameterizations and previous studies.
kw CHBr3 CH2Br2 CHClBr3
GOMECC Open Ocean S07 28.5 (-8.7 - 126.1)
6.6 (-0.5 - 26.0)
0.5 (-1.2 - 3.0)
N00 26.7 (-8.3 - 126.9)
6.2 (-0.4 - 26.0)
0.5 (-1.2 - 2.9)
W92 38.5 (-11.9 - 183.3)
9.0 (-0.6 - 37.6)
0.7 (-1.7 - 4.2)
GOMECC Coastal South of 40oN S07 45.7 (-25.4 - 334.3)
11.6 (1.0 - 62.0)
1.0 (-0.4 - 5.4)
N00 44.1 (-24.0 - 310.9)
11.3 (0.8 - 58.4)
1.0 (-0.3 - 5.1)
W92 63.7 (-34.6 - 449.1)
16.3 (1.2 - 84.4)
1.4 (-0.5 - 7.3)
GOMECC Coastal North of 40oN S07 85.1 (0.6 - 1056.3)
11.2 (0.3 - 112.3)
1.0 (-0.3 - 10.8)
N00 72.8 (0.2 - 909.5)
9.5 (0.1 - 98.4)
0.8 (-0.2 - 9.3)
W92 105.2 (0.3 - 1313.7)
13.7 (0.1 - 142.2)
1.2 (-0.3 - 13.5)
Jones et al. [2009] (Macroalgae dominant coast, Sept 2006) N00 mean 1800 mean 18.0 -
Zhou et al. [2005] (Great Bay, NH, Aug 2003) - 624.0 ± 1368.0 112.8 ± 129.6 -
Zhou et al. [2005] (Gulf of Maine, Aug 2002) W92 median 465.6
(33.6 – 4392.0) - -
Zhou et al. [2008] (Coastal region of New Hampshire, summer 2002 - 2004) - 453.6 ± 295.2 62.4 ± 45.6 -
Hughes et al. [2009] (Antarctica, summer 2006/2007) N00 84.0
(-13.0 - 275.0) 21.0
(2.0- 70.0) -
Carpenter et al. [2009] (Tropical Atlantic Coastal, summer 2006 and 2007) N00 236.0
(127.0 - 322.0) 72.2
(62.7 - 84.5) -
As shown in Table 2-5, coastal net fluxes differ significantly between
locations, even when comparisons were made in the same season to attempt to
eliminate the effects of seasonality. This variability is likely due to spatial and
temporal variations in productivity, coastal mixing, factors that affect gas exchange,
such as temperature and wind speed. Variations in the parameterization of the gas
exchange coefficient are also important. Moreover, inter study comparisons also need
to be treated with caution. The subsets of coastal sea-to-air fluxes of CHBr3 and
CH2Br2 measured during the GOMECC cruise are lower than those observed in other
coastal regions. This is partly attributable to the differences in coastal coverage. Most
28
of the referenced studies were conducted in relatively confined coastal regions.
Therefore, the source types of VSLSs should be relatively constant for the duration of
each of those studies. The GOMECC cruise covered more than 5000 km of coastline.
The sampling time and data density for each environmental regime encountered during
the GOMECC cruise varied substantially. Therefore, global coastal extrapolations
using limited coastal data which may be dominated by certain sources may not be
appropriate. The extrapolated global coastal net fluxes for the VSLSs using the
GOMECC cruise values are not presented in this paper, as the coastal environmental
regimes in this region may not be globally representative.
2.3.5 Calculated production rates
Production rates (P, nmol m-3 d-1) (Table 2-3, Figure 2-5c) are calculated as
described by Yvon-Lewis et al. [2004], assuming steady state, where mixed layer
production equals total mixed layer loss:
P = !w!(!"!!"cfc-11)!a
!""!!
+ kloss(𝐶𝐶w) (Equation 2.3)
kloss = kh + Jz + khs for CHBr3;
kloss = kh + kB for CH2Br2;
kloss = kh for CHClBr2;
where SAcfc-11 (%) is the saturation anomaly for CFC-11; kloss (d-1) is the total pseudo
first order loss rate constant for all the possible loss terms; kh (d-1) is the hydrolysis
rate constant [Mabey and Mill, 1978; Washington, 1995]; khs (d-1) is the halide
substitution pseudo first order rate constant [Geen, 1992]; Jz (d-1) is the photolysis loss
29
rate constant in the mixed layer; and kB (d-1) is the pseudo first order biological
degradation rate constant for CH2Br2 [Goodwin et al., 1998]. The chlorofluorocarbons
(CFCs) are conservative tracers, and the SAcfc-11 correction accounts for any physical
forcing (e.g. warming or cooling of surface seawater) that may create disequilibrium
between the ocean and the atmosphere. The calculated production rates should be
interpreted only as “apparent” production rates. Some of the apparent production may
be the result of water mass transport in the complex coastal environment that is not
captured by variations in CFC concentrations.
The calculated production rates for the entire GOMECC cruise for CHBr3,
CH2Br2, and CHClBr2 are 10.0 (-2.6 to 395.0) nmol m-3 d-1, 6.2 (1.1 to 101.7) nmol m-
3 d-1, and 0.1 (-0.1 to 3.6) nmol m-3 d-1, respectively. The coastal “hot spot” for VSLSs
production during the GOMECC cruise is located north of 40oN (Table 2-3, Figure 2-
5c), which may be due to existence of macroalgae or proximity to the Boston outfall.
These rates are meant to provide some general idea of production rate distributions of
the VSLSs with the assumption of steady state.
2.4 Conclusion
The GOMECC cruise data provided CHBr3, CH2Br2, and CHClBr2
concentration, saturation anomaly, flux and production rate distributions in an
extensively urbanized coastal area. These compounds were supersaturated almost
everywhere in the study region, indicating waters of the east coast of United States are
sources of the VSLSs to the atmosphere. Significant correlations between the VSLS
30
seawater concentrations suggest they could share common sources in our study area
during the cruise. Maximum values for seawater concentrations, saturation anomalies,
fluxes and calculated production rates for the VSLSs are observed north of 40°N,
where human influences and macroalgal production are implicated. CHBr3 seawater
concentrations and coastal net fluxes in this region were significantly higher than in
the area south of 40°N, where the co-existence of phytoplankton and anthropogenic
sources is implicated.
These results suggest a few possibilities. First, anthropogenic input such as
treated water outfalls and nuclear power plants may only have a significant impact on
the local scale. Second, the phytoplankton species presented during this study may not
be prolific sources of VSLSs. Third, the maxima observed in the north are partly the
result of macroalgal sources.
While the saturation anomalies suggest that the gulf and the eastern coasts of
the United States are sources of VSLSs to the atmosphere, both the CHBr3 and
CH2Br2 mean coastal net fluxes lie at the low ends of the ranges of fluxes that were
observed in other regions. However, these differences could be due to the different
spatial resolutions of the various studies. The photolytic loss of CHBr3, under the
conditions observed during this study, is not negligible and should be considered in
future studies of CHBr3. Environmental parameters such as chlorophyll a, CDOM, and
CH2Br2 to CHBr3 concentration ratio characteristics observed from other natural
environments are not good proxies for determining the sources of these VSLSs during
the GOMECC cruise. More work is needed in order to distinguish VSLS contributions
from different sources in complex urbanized coastal environments. Since coastal
31
environments are complicated and many features are regionally specific, global
extrapolation should be treated with caution.
32
CHAPTER III
SPATIAL DISTRIBUTION OF BROMINATED VERY SHORT-LIVED
SUBSTANCES IN THE EASTERN PACIFIC*
3.1 Introduction
Bromoform (CHBr3), dibromomethane (CH2Br2), bromodichloromethane
(CHBrCl2), chlorodibromomethane (CHClBr2), along with bromochloromethane
(CH2BrCl), are the most abundant brominated very short-lived substances (BrVSLS)
in the atmosphere [Montzka and Reimann, 2011]. These compounds are important
sources of inorganic bromine (Bry) to the troposphere and lower stratosphere, in
addition to the longer-lived compounds such as halons and methyl bromide. Thus,
these BrVSLS play an important role in catalytic ozone destruction in the atmosphere.
BrVSLS mainly originate from natural sources in seawater and enter the marine
boundary layer via sea-to-air fluxes [Quack and Wallace, 2003]. Anthropogenic
sources, such as seawater-cooled power plants, also contribute to elevated
concentrations of CHBr3, CHClBr2, and CHBrCl2 observed in coastal waters;
however, these sources may only be significant on a local scale [Quack and Wallace,
2003].
Elevated concentrations of brominated BrVSLS in seawater and the adjacent
atmosphere are thought to be affected by macroalgae and phytoplankton production
[Carpenter and Liss, 2000; Goodwin et al., 1997; Karlsson et al., 2008; Manley et al., * Reprinted with permission from “Spatial distribution of brominated very short-lived substances in the Eastern Pacific” by Y. Liu et al., 2013. Journal of Geophysical Research, Oceans, DOI: 10.1002/jgrc.20183., Copyright [2013] by John Wiley and Sons.
33
1992; Moore et al., 1996; Moore et al., 1995b; Quack et al., 2007a; Sturges et al.,
1992; Sturges et al., 1993]. For example, elevated BrVSLS concentrations in surface
seawater have been observed near regions where surface chlorophyll a concentrations
were also elevated [Carpenter et al., 2009; Schall et al., 1997]. Although
phytoplankton production has been suggested as a possible source, poor correlation
between chlorophyll a and BrVSLS concentrations observed in different regions have
raised questions regarding the sources and mechanisms of formation and degradation
of these compounds in seawater. Therefore, many uncertainties remain concerning
environmental factors that influence production and distributions of BrVSLS and the
role of phytoplankton in BrVSLS production.
The enzyme bromoperoxidase is thought to be responsible for the production
of some poly-brominated compounds, such as CHBr3 and CH2Br2, in the presence of
hydrogen peroxide (H2O2) and dissolved organic matter (DOM) as the substrate [Hill
and Manley, 2009; Lin and Manley, 2012; Manley, 2002; Manley and Barbero, 2001;
Moore et al., 1996; Theiler et al., 1978; Wever et al., 1993]. The presence of
bromoperoxidase in macroalgae and microalgae appears to be class and species-
specific, which may partly explain the observed weak correlations between BrVSLS
and chlorophyll a concentrations. Several studies have used pigment biomarkers to
identify specific taxa of phytoplankton that may be significant BrVSLS sources in
seawater [Abrahamsson et al., 2004; Karlsson et al., 2008; Mattson et al., 2012;
Quack et al., 2007a; Raimund et al., 2011; Roy, 2010]. For example, Quack et al.
[2007a] showed significant correlations between CHBr3 seawater concentrations with
the abundances of cyanobacteria (Synechococcus spp.) and diatoms in the Mauritanian
34
upwelling region. Karlsson et al. [2008] also suggested that some species of
cyanobacteria in the Baltic Sea were possible sources of CHBr3. Other phytoplankton
groups, such as prymnesiophytes, green algae, and prochlorophytes, may have also
contributed to the production of BrVSLS, as observed by Raimund et al. [2011] near
the Iberian upwelling system. However, in certain regions, such as the Antarctic,
elevated diatom abundances may, in part, have contributed to the observed brominated
BrVSLS concentrations in seawater; yet, no significant correlations were found
between the BrVSLS and phytoplankton pigments [Abrahamsson et al., 2004; Mattson
et al., 2012].
In this study, depth-profiles of CHBr3 (219 samples), CH2Br2 (236 samples),
CHClBr2 (238 samples), and CHBrCl2 (238 samples) were measured during the
Halocarbon Air-Sea Transect – Pacific (HalocAST-P) cruise. Phytoplankton pigment
concentrations, picoplankton abundance, and inorganic nutrient concentrations were
measured to determine relationships between phytoplankton abundance and BrVSLS
concentrations over the large spatial range covered by this cruise.
35
Figure 3-1. Sampling station map for the Halocarbon Air-Sea Transect – Pacific cruise, superimposed on March – April 2010 monthly averaged SeaWiFs chlorophyll a concentrations [Acker and Leptoukh, 2007].
3.2 Sampling and analyses methods
The Halocarbon Air-Sea Transect – Pacific (HalocAST-P) cruise, conducted
onboard the R/V Thomas G. Thomson, departed from Punta Arenas, Chile on March
31, 2010 and arrived in Seattle, WA, United States on April 28, 2010 (Figure 3-1).
The cruise track was designed to observe oceanic and atmospheric latitudinal
distributions of a suite of halocarbon compounds including CHBr3, CH2Br2, CHBrCl2,
and CHClBr2. A total of 24 CTD/rosette casts were conducted along the cruise track
(one cast per day at local noon) and water was collected from 12 depths ranging from
36
~5 m to 750 m. Here onwards, we refer the “surface” values to measurements made at
~5 m. For stations with water depths less than 750 m, i.e. casts 1, 2, and 3, the water
columns were sampled from the surface to just above the sediment. Data collected
from casts 1 and 2, where water depths were less than 200 m, are considered as coastal
data. These definitions are approximately equivalent to “coastal and coastally
influenced waters” defined by Carpenter et al. [2009] and “shelf regime” defined by
Quack et al. [2004]. Cast 3 with water depth less than 400 m, although not coastal by
our definition, was located on the continental shelf. Seawater samples were collected
using a CTD/rosette sampler with 24 10-L Niskin bottles. The CTD/rosette package
was also equipped with an oxygen sensor, a fluorescence sensor, and a
transmissometer.
3.2.1 Halocarbons
Samples for halocarbon analysis were collected using 100 mL gas tight ground
glass syringes immediately after the rosette unit was on deck. The samples were
immediately transferred to a purge-and-trap sample storage module. The purge-and-
trap sample storage module is a compact thermoelectric refrigerator held at ~ 6 ºC.
Inside, sixteen 70 mL glass bulbs were connected to a 16-position loop selection valve
(Valco Instrument Co.), which was connected to the purge-and-trap system. This
configuration allowed seawater samples to be stored in a cold, dark and gas tight
environment until analysis [Yvon-Lewis et al., 2004]. Each seawater sample was
37
gently injected into the appropriate bulb through a 0.2 µm membrane filter to remove
microorganisms. All of the samples were analyzed within 12 hours of collection.
For gas analysis, each seawater sample in the glass bulb was sequentially
transferred into a glass sparger with an ultra-pure helium gas stream, and purged at
144 mL min-1 at 40ºC. The whole sample line was then flushed with the same ultra-
pure helium gas stream until it was time for the next sample to ensure no residue from
the previous sample was left in the line. The sample purge stream was dried with two
in-line Nafion driers (Perma Pure, NJ), and the analytes were pre-concentrated on one
cryotrap and focused on a second cryotrap that were both held at -75°C, and then
desorbed at 200ºC. The analytes were desorbed from the focusing trap and transfered
by chromatography grade helium carrier gas into the gas chromatograph – mass
spectrometer (GC-MS) for quantification (Agilent GC 6890N and MS 5973N; DB-
VRX 0.180 ID column with 10 m pre-column and 30 m main column). Detailed
analytical method was described in Yvon-Lewis et al. [2004] and references therein.
Moist whole air gas calibration standards used in the field were calibrated
before and after the cruise against gas standards obtained from the National Oceanic
and Atmospheric Administration Global Monitoring Division (NOAA GMD). The
field calibration standard was run after every fourth injection. The ultra-pure helium
gas stream was run as a blank after every three samples to monitor flushing efficiency.
The detection limit for CHBr3 and CH2Br2 were 1.00 × 10-3 pmol L-1. For CHClBr2
and CHBrCl2, the detection limits were 5.00 × 10-3 pmol L-1. The averaged analytical
uncertainty was 7.00 % for CHBr3, 6.00 % for CH2Br2 , and 9.00 % for CHClBr2 and
CHBrCl2. Purge efficiency was determined by re-stripping seawater samples three
38
times, and the percentage in total concentration for the first stripping was 72.3 % for
CHBr3, 72.4 % for CH2Br2, 77.0 % for CHClBr2, and 93.7 % for CHBrCl2. BrVSLS
concentrations reported here were corrected for purge efficiencies.
3.2.2 Pigment, picoplankton counts, and nutrients
Pigment samples were collected from designated Niskin bottles at 5 m, the
chlorophyll maximum depth, and at 750 m. For each cast, 5 to 10 L of seawater at
each depth were filtered through a pre-combusted 0.7 µm nominal pore size GF/F
filter until color was visible on the filter. The filters were frozen immediately at -80 °C
and kept frozen until analysis back in the laboratory. The phytoplankton pigment
extraction procedure was followed as described in Wright et al. [1991]. Briefly, filters
were extracted ultrasonically with acetone, centrifuged, and the supernatant blown to
dryness under a nitrogen stream at room temperature in the dark. The concentrated
residue was re-dissolved in 150 µL of acetone prior to the analysis by high-
performance liquid chromatography (HPLC). Pigments were analyzed using a Waters
HPLC coupled to an on-line 996 photodiode array detector (PDA), a fluorescence
detector (Shimadzu RF535) (ex: 440 nm; em: 660 nm), on a reversed-phase Grace
Adsorbosphere C18 column (5 µm, 250 mm x 4.6 mm i.d.), using the gradient flow
described by Chen et al. [2003]. A total of 18 pigment biomarkers were analyzed,
which included total chlorophyll a (chlorophyll a + divinylchlorophyll a), chlorophyll
b, c2 and c3, total carotene (α + β), peridinin, 19-butanoyloxyfucoxanthin, fucoxanthin,
19-hexanoyloxyfucoxanthin, prasinoxanthin, pheophorbide a, violaxanthin,
39
diadinoxanthin, alloxanthin, diatoxanthin, lutein, pheophytin a, and zeaxanthin.
Pigment standards were purchased from DHI Water and Environment, Denmark. The
pigment standards were run individually and as a mixed standard to determine
retention times, spectra, and the response factors.
The pigment biomarker zeaxanthin can provide information about the presence
of cyanobacteria [Jeffrey et al., 2011; Wright and Jeffrey, 2006]. However, this
pigment is common in both Prochlorococcus and Synechococcus, two genera of
cyanobacteria that often dominate the picoplankton. To gain specific information on
the potential picoplankton contribution to the BrVSLS seawater concentrations, these
two genera were distinguished and enumerated using flow cytometry. In addition, flow
cytometry counts were used to provide abundance information on picoeukaryotes (< 3
µm algae including chlorophytes, pelagophytes, and haptophytes) and heterotrophic
bacteria. Picoplankton samples were collected from the Niskin bottles, 1 mL of the
sample was filtered through a 35 µm mesh cell strainer and fixed with 20 µL 10%
paraformaldehyde for 10 minutes (final concentration 0.2%) and then quickly frozen
at -80 °C and kept frozen until analysis. Enumeration of picoplankton and
heterotrophic bacteria was performed using a Becton Dickinson FACSCalibur flow
cytometer [Campbell, 2001]. For the non-pigmented heterotrophic bacteria, the cells
were stained with SYBR Green (Molecular Probes) prior to introduction into the flow
cytometer. Data were analyzed using CytoWIN [Vaulot, 1989].
Inorganic nutrient samples, nitrate (NO3-), nitrite (NO2
-), orthophosphate
(HPO42-), and silicate (SiO4
4-), were sent to the Geochemical and Environmental
Research Group (GERG) at Texas A & M University for analysis. Inorganic nutrient
40
concentrations were determined using the Astoria-Pacific auto-analyzer following the
methodologies of Armstrong et al. [1967] and Bernhardt and Wilhelms [1967].
3.2.3 Data handling and statistical methods
All data were checked for quality. Exceptionally high or low concentrations
measured due to occasional instrumental error, which were determined from CFC-11
concentration as an inert tracer in the same sample, were removed. Subsequently, the
data were tested for normality to determine the most appropriate statistical method
used for data interpretation. Spearman’s rank correlation was chosen to assess the
association between parameters, due to the non-normal distributions of the data.
Spearman’s rank correlation analyses were evaluated at 95% confidence level.
Significance of the correlation was determined using a two-tailed test. Significant
differences in means between data groups were determined by one-way ANOVA test
at 95% confidence level. All statistical analyses were performed using the OriginLab®
version 9.0 statistic module.
3.3 Results and discussion
3.3.1 Hydrography and water column layers during the HalocAST-P cruise
Based on profiles of salinity, temperature, potential density (σθ), dissolved
oxygen, and CFC-11 (Figures 3-2, 3-3 and 3-4), there were four main hydrographic
41
features observed during the HalocAST-P cruise: (1) freshwater discharged from the
coasts, (2) the entrainment of surface waters into deeper water column, (3) the
Antarctic intermediate water (AAIW) in the southern hemisphere, and (4) the North
Pacific intermediate water (NPIW) in the northern hemisphere. There was apparent
entrainment of surface waters into the deeper water column, which was probably due
to seasonal fluctuation of thermocline depths and mixing. Based on the dissolved
oxygen profile, surface water entrainment reached down to ~ 300 m in the southern
temperate waters and to ~ 500 m in the northern temperate waters. Lower salinity and
elevated CFC-11 concentrations were also observed in these regions. The AAIW is
characterized as low salinity (~34.2) [Pickard, 1990] located at 26.98 < σθ < 27.80
isopycnal [Schmidtko and Johnson, 2011] in the southern hemisphere, which was ~
500 m and below in the temperate waters during the HalocAST-P cruise (Figure 3-2,
and 3 - 3a). Near the Chilean shelf, the top of AAIW can be as shallow as ~ 300 m
[Palma et al., 2005]. The AAIW is also characterized as enriched in NO3- and HPO4
2-
and low in HSiO3- concentrations (Figure 3-5) [Sarmiento and Gruber, 2006], and
enriched in oxygen and CFC-11 [Hartin et al., 2011] (Figure 3-4). The NPIW is
characterized as low salinity located at 26.60 < σθ < 27.40 isopynal [Talley, 1997] in
the north Pacific, which was ~ 300 m and below in the temperate waters during the
HalocAST-P cruise (Figure 3-2 and 3-3b). This water mass is enriched in nutrients
such as NO3-, HPO4
2-, and HSiO3- [Sarmiento and Gruber, 2006] (Figure 3-5), and
depleted in CFC-11, with an apparent age of a few decades in the eastern boundary of
the Pacific ocean [Watanabe et al., 1994] (Figure 3-4).
42
To better understand BrVSLS distributions in the water column, the water
column was divided into three layers: mixed layer, below mixed layer within euphotic
zone, and below euphotic zone. The bottom of the mixed layer was defined following
Brainerd and Gregg [1995]. The bottom of the euphotic was defined as depths where
photosynthetic active radiation (PAR) is 1% of surface value, as measured at ~ 5 m
[Kirk, 1994].
Figure 3-2. Salinity profile for the HalocAST-P cruise.
43
Figure 3-3. Temperature-salinity (TS) diagram. (a) Below ~ 500 m of casts 4 (red), 5 (green), 6 (blue), and 7 (magenta) were the Antarctic intermediate water (AAIW; marked with black square). Casts 2 and 8 were plotted in grey lines as reference of water masses not in the AAIW. (b) Below ~300 m of casts 21(red), 22 (green), 23 (blue), and 24 (magenta) were the North Pacific intermediate water (NPIW; marked with black square). Casts 22 and 25 (TS only, no BrVSLS data collected for Cast 25, conducted in Puget Sound, Seattle, USA) were plotted in grey lines as reference of water masses not in the NPIW.
44
Figure 3-4. (a) Dissolved oxygen profile and (b) CFC-11 profile during the HalocAST-P cruise.
45
Figure 3-5. (a) Silicate (HSiO3-), (b) orthophosphate (HPO4
2-), and (c) nitrate (NO3-)
profile during the HalocAST-P cruise.
46
3.3.2 Spatial distributions of CHBr3, CH2Br2, CHClBr2, and CHBrCl2
In the mixed layer, CHBr3, CH2Br2, CHClBr2, and CHBrCl2 concentrations
were in general higher in the coastal ocean (depth ≤ 200 m) and the tropics (~ 20° S to
20° N) (Table 3-1; Figure 3-6). Such a general trend is consistent with that observed in
other regions [Butler et al., 2007; Carpenter et al., 2007b; Class and Ballschmiter,
1988; Liu et al., 2011; Quack and Wallace, 2003]. CHBr3 and CH2Br2 concentrations
were higher below the mixed layer within the euphotic zone than those observed in the
mixed layer and below euphotic zone (Table 3-1; Figures 3-7 and 3-8). The maximum
seawater concentrations for CHBr3, CH2Br2, and CHClBr2 were observed near the
depths of subsurface chlorophyll a maxima in subtropical waters (Table 3-1; Figure 3-
7), which suggests that the production of these compounds is likely related to
photosynthetic biomass production and associated biochemical processes. Such
features were consistent with the report by Quack et al. [2004], who also found that
CHBr3 concentration maxima in the Atlantic tropical ocean were located below the
mixed layer, near the chlorophyll a maxima. The authors reported subsurface CHBr3
concentration maxima ranged from 14 to 60 pmol L-1. Subsurface CHBr3
concentration maxima in the Pacific tropical ocean (~ 20° S to 20° N) were within the
range reported by Quack et al. [2004] (Figure 3-7). Patches of elevated CHBr3,
CH2Br2, and CHClBr2 below the bottom of tropical euphotic zone (~ 20° S to 20° N)
were likely due to entrainment of water from the euphotic zone, where high
concentrations of BrVSLS were produced.
47
Table 3-1. Mean (range; number of samples) BrVSLS concentrations (pmol L-1) in coastal and open ocean water column. Bold text highlighted the minimum and maximum of the data.
CHBr3 CH2Br2 CHClBr2 CHBrCl2 Coastal ocean
Mixed Layer 17.46
(13.80 - 20.67; 13) 4.79
(3.36 - 6.51; 13) 2.59
(2.00 - 3.11; 13) 1.69
(1.28 - 2.10; 13) Below Mixed layer, within euphotic zone
-
2.19 (1.78 - 2.59; 2)
0.29 (0.10 - 0.49; 2)
0.15 (0.11 - 0.19; 2)
Below euphotic zone
- 1.79
(1.62 - 1.96; 2) 0.49
(0.46 - 0.52; 2) 0.31
(0.11 - 0.52; 2) Open ocean
Mixed Layer 2.47
(0.15 - 11.32; 65) 1.61
(0.70 - 3.91; 69) 0.51
(0.14 - 1.39; 69) 0.24
(0.01 - 0.95; 69) Below Mixed layer, within euphotic zone
6.32 (0.08 - 31.77; 35)
6.00 (1.25 - 24.97; 37)
1.11 (0.29 - 3.72; 37)
0.40 (0.04 - 2.26; 37)
Below euphotic zone 2.78
(0.01 - 19.44; 106) 1.99
(0.04 - 17.23; 113) 0.63
(0.05 - 2.76; 115) 0.64
(0.03 - 4.23; 115)
48
Figure 3-6. Latitudinal distributions of mean mixed layer CHBr3, CH2Br2, CHClBr2, and CHBrCl2 concentrations, error bars indicate ±1 standard deviation of the mixed layer concentrations.
49
Figure 3-7. Depth profiles of (a) CHBr3, (b) CH2Br2, (c) CHClBr2, (d) CHBrCl2 and (e) CHCl3. White line marks the bottom of mixed layer, black line marks the depths of chlorophyll a maxima, and red line marks the bottom of the euphotic zone.
50
Figure 3-8. Boxplots of data grouped based on geographical setting (open ocean vs. coastal ocean), and data grouped based on water column layers (mixed layer, below mixed layer within euphotic zone, and below euphotic zone), for CHBr3 (a and b), CH2Br2 (c and d), CHClBr2 (e and f), and CHBrCl2 (g and h). Vertical line in the box indicates median of data, filled square indicates mean of data, box range indicates 25th to 75th percentile of data, whisker indicates 10th to 90th percentile of data, open box indicates 5th to 95th percentile of data. Stars indicate minimum and maximum of data. Note that the y-axis is a log-scale.
51
CHClBr2 and CHBrCl2 concentrations were higher below the euphotic zone
than in the mixed layer (Figures 3-7 and 3-8), which suggest there were sources of
these two BrVSLS in deeper waters. In fact, the CHBrCl2 concentration range below
the euphotic zone was even higher than near the chlorophyll a maxima. CHBrCl2
concentration was greatest below the euphotic zone in the northern temperate water
column (Figure 3-7), which suggests source strengths of CHBrCl2 in deeper waters
could be higher than in the euphotic zone in certain regions (Figure 3-8). Our results
were consistent with the observations of Moore and Tokarczyk [1993] in the
Northwest Atlantic Ocean, where CHClBr2 and CHBrCl2 concentrations were higher
in deeper waters compared to surface waters. The authors suggested slow successive
CHBr3 chlorine substitutions to CHClBr2, then CHBrCl2 [Class and Ballschmiter,
1988; Geen, 1992], may in part explained such features, as CHBr3 produced at the
upper water columns mixed and/or diffused into deeper waters.
Patches of elevated CHBr3, CHClBr2, and CHBrCl2 were observed below the
euphotic zone, particularly in the AAIW and NPIW (Figure 3-7). Other studies have
reported high concentrations of BrVSLS in surface Antarctic waters. For example,
concentration ranges from 5.25 to 280 pmol L-1 and 2.99 to 30.0 pmol L-1 were
observed in Antarctic surface seawater for CHBr3 and CH2Br2, respectively,
depending on the location of observations [Abrahamsson et al., 2004; Butler et al.,
2007; Carpenter et al., 2007a; Hughes et al., 2011; Mattson et al., 2012]. As
suggested by these studies, the high CHBr3 and CH2Br2 concentrations may be
attributed to sources like phytoplankton blooms and sea ice. Hydrolysis half-lives of
CHBr3, CH2Br2, CHClBr2, and CHBrCl2 at 20°C are 686, 183, 137, and 274 years,
52
respectively [Vogel et al., 1987]. Chlorine substitution half-life of CHBr3 is ~ 5 to 74
years depending on seawater temperature [Geen, 1992]. With such long half-lives of
these BrVSLS in seawater, it is not surprising that low concentrations of these
BrVSLS were retained in the AAIW, as the water mass subsided from the surface
where prolific sources of these BrVSLS existed. While elevated CHClBr2 and
CHBrCl2 concentrations in the AAIW may be partially attributed to successive
chlorine substitution of CHBr3, it is difficult to assess the magnitude of the
contributions of this process to the features observed in this study.
Nonetheless, it would be reasonable to speculate that the CHBrCl2 maxima and
elevated CHClBr2 located in the NPIW were, at least partially, the result of successive
chlorine substitution of CHBr3. The apparent age of NPIW (few decades) in this
location was old enough relative to chlorine substation half-life of CHBr3 to result in
such a feature. In this water mass, CHBr3 concentrations were not elevated, which may
potentially suggest conversions to CHClBr2 and then CHBrCl2. Although chloroform
(CHCl3) is not discussed in detail in this study, it is worth noting that CHCl3
concentrations were also elevated in the NPIW (Figure 3-7e), probably due to further
chlorine substitution of CHBrCl2. Such features of CHCl3 in NPIW further support the
hypothesis of successive chlorine substitution of CHBr3.
53
3.3.3 Sources of CHBr3, CH2Br2, CHClBr2, and CHBrCl2 in the euphotic zone
3.3.3.1 Sources of CHBr3, CH2Br2, CHClBr2, and CHBrCl2 in the coast
Elevated CHBr3, CH2Br2, CHClBr2, and CHBrCl2 concentrations were
observed in Chilean coastal waters (i.e. Cast 1 and 2; Figures 3-6, 3-7, and 3-8).
Distinctly lower salinity in this region implies freshwater input from the continent
(Figure 3-2). The southern Chilean coast, where Cast 1 and 2 were collected, is
characterized as a fjord coast, dominated by islands, channels, and fjords [Fariña et
al., 2008]. The subtidal kelp Macrocystis pyryfera and the intertidal kelp Durvillea
antarctica are the most representative macroalgae in this region [Fariña et al., 2008;
Lüning, 1990; Ríos et al., 2007]. In this region, phytoplankton assemblages have
heterogeneous spatial and temporal distribution patterns, with diatoms being the most
abundant phytoplankton group year-round [Alves-de-Souza et al., 2008]. The giant
kelp Macrocystis pyryfera has been shown to produce considerable amounts of CHBr3
and CH2Br2 [Manley et al., 1992]. Numerous laboratory studies also showed
production of CHBr3 and CH2Br2 from certain diatoms, which are likely associated
with bromoperoxidase activity [Hill and Manley, 2009; Moore et al., 1996; Moore et
al., 1995b; Tokarczyk and Moore, 1994]. Therefore, it is not surprising that the
maximum mixed layer concentrations of CHBr3, CH2Br2, CHClBr2, and CHBrCl2
were observed in this region (Table 3-1, Figure 3-6), which may be explained either
due to in situ biologically mediated production or transport from the channels.
54
During the BLAST-I cruise conducted in 1994, high CHBr3 and CH2Br2
concentrations, up to 90.23 and 25.83 pmol L-1, respectively, were observed in the
inland passage [Butler et al., 2007], which supports the role of channels on the Chilean
coast as significant sources of BrVSLS to the atmosphere. The HalocAST-P cruise
track followed the coast outside of the inland passage and mixed layer CHBr3 and
CH2Br2 concentrations were much lower than those observed during the BLAST I
cruise (Table 3-1, Figure 3-6). Such a substantial differences in CHBr3 and CH2Br2
concentrations may suggest significant spatial variability in their concentrations, if we
assume inter-annual differences in CHBr3 and CH2Br2 production was not significant.
The fact that CHBr3 and CH2Br2 concentrations were higher inside the inland passage
may suggest more prolific sources. Moreover, unlike outside the inland passage, the
restricted water circulations in the inland passage may also allow CHBr3 and CH2Br2
to accumulate. Despite the fact that the CHBr3 and CH2Br2 mixed layer concentration
maxima observed in this study were lower than other studies in macroalgal-dominated
regions, where extremely high concentrations of BrVSLS were observed in surface
waters above macroalgal beds, the concentration patterns observed in the Chilean
coast were consistent with other studies [Carpenter and Liss, 2000; Fogelqvist and
Krysell, 1991; Goodwin et al., 1997; Gschwend and MacFarlane, 1986; Jones and
Carpenter, 2005; Laturnus, 1996, 2001; Laturnus et al., 1996; Liu et al., 2011; Moore
and Tokarczyk, 1993; Yokouchi et al., 2005]. Lin and Manley [2012] found that CHBr3
and CH2Br2 production is a function of coastal dissolved organic matter (DOM)
characteristics (i.e. size fraction and source). Therefore, terrestrial-derived DOM
55
carried by the channels may also influence CHBr3 and CH2Br2 concentrations
observed in this region.
Flow cytometry cell counts of picoplankton, which have the same sampling
resolution as the BrVSLS, allowed us to examine BrVSLS biological sources in
further detail. In the Chilean coastal region, CH2Br2 was significantly correlated with
Synechococcus spp. and picoeukaryotes (< 3 µm algae including chlorophytes,
pelagophytes, haptophytes), and CHClBr2 was significantly correlated with
heterotrophic bacteria (Table 3-2). However, CHBr3 and CHBrCl2 did not show any
association with any of the picoplankton groups. Relationship between BrVSLS with
phytoplankton taxonomic group derived from pigment analysis was not considered in
the coastal region due to small sample size (n = 5).
56
Table 3-2. Spearman’s rank correlation coefficient (ρ) of BrVSLS with picoplankton, p-value and number of samples (n) are presented in the parentheses. Relationships between the BrVSLS with photosynthetic picoplanktons were not considered below the euphotic zone. Below the euphotic zone, only relationships between BrVSLS and heterotrophic bacteria were considered. “ns” indicates not significant correlation.
3.3.3.2 Sources of CHBr3, CH2Br2, CHClBr2, and CHBrCl2 in the open ocean
In the surface open ocean, CH2Br2 was significantly correlated with 19’-
hexanoyloxyfucoxanthin (ρ = 0.52, p = 0.03, n = 17), which is the pigment biomarker
for prymnesiophytes [Jeffrey et al., 2011; Wright and Jeffrey, 2006]. In the open ocean
chlorophyll a maximum depths, CHClBr2 was significantly correlated with
violaxanthin (ρ = 0.54, p = 0.03, n = 19), which is the pigment biomarker for green
Heterotrophic
bacteria Prochlorococcus Synechococcus Picoeukaryotes Coastal euphotic zone
CHBr3 ns ns ns ns
CH2Br2 ns ns 0.92
(<< 0.001; 13) 0.90
(<< 0.001; 13)
CHClBr2 0.59
(<< 0.03; 13) ns ns ns CHBrCl2 ns ns ns ns
Open Ocean Mixed Layer
CHBr3 ns ns 0.27
(0.03; 65) ns
CH2Br2 0.36
(<< 0.003; 65) -0.25
(0.04; 67) 0.46
(<< 0.001; 68) ns
CHClBr2 0.24
(<< 0.05; 65) ns 0.27
(0.02; 68) ns CHBrCl2 ns ns ns ns
Below Mixed layer, within euphotic zone CHBr3 ns ns ns ns
CH2Br2 ns ns ns ns
CHClBr2 ns 0.36
(0.03; 36) ns ns
CHBrCl2 ns ns ns 0.37
(0.03; 36) Below euphotic zone
CHBr3 ns - - -
CH2Br2 0.29
(0.003; 109) - - - CHClBr2 ns - - -
CHBrCl2 ns - - -
57
algae [Jeffrey et al., 2011; Wright and Jeffrey, 2006]. CHBr3 and CHBrCl2 were not
significantly correlated with any of the pigment biomarkers. While some studies have
observed correlations between CHBr3 and pigment biomarkers [Quack et al., 2007a;
Raimund et al., 2011], the lack of correlation between CHBr3 and pigment biomarkers
is not uncommon. Abrahamsson et al. [2004] and Mattson et al. [2012] consistently
found that CHBr3 did not yield significant correlations with any of the pigment
biomarkers in Antarctic seawater. Such a lack of correlation between CHBr3 and
pigment biomarkers may be due to differences in their turnover times in the water
column [Mattson et al., 2012], short term (diurnal) variability in CHBr3 production
rate [Ekdahl et al., 1998], species specificity to CHBr3 production (which cannot be
resolved by using pigment biomarkers), and/or the interplay between different
phytoplankton and bacterioplankton groups.
Zeaxanthin is a pigment biomarker commonly used for assessing the presence
of cyanobacteria [Jeffrey et al., 2011; Wright and Jeffrey, 2006]. However, unlike the
flow cytometry data, information from zeaxanthin distributions cannot distinguish
important cyanobacteria taxa such as Synechococcus and Prochlorococcus. Several
studies have indicated cyanobacteria as a possible source of CHBr3 [Karlsson et al.,
2008; Quack et al., 2007a]. Therefore, data from flow cytometry were employed to
examine the relationship between the BrVSLS and picoplankton in greater detail. In
addition, heterotrophic bacteria counts were also obtained, which allowed us to
examine the potential influence of heterotrophic bacteria in BrVSLS biogeochemistry.
However, it should be noted that heterotrophic bacteria abundance does not
necessarily correlate with bacterial activity. We found that the BrVSLS were
58
correlated with various picoplankton groups at different layers of the water column
(Table 3-2). For example, CHBr3, CH2Br2, and CHBrCl2 were significantly correlated
with Synechococcus spp. in the open ocean mixed layer (Table 3-2). Despite the fact
that CHBr3 was only weakly correlated with Synechococcus, more detailed
phytoplankton assemblage analyses could reveal relationships between CHBr3 and
specific groups of phytoplankton.
CH2Br2 was significantly correlated with heterotrophic bacteria in the mixed
layer and below the euphotic zone (Table 3-2), which may indicate bacterial
production. While bacterial degradation of CH2Br2 has been observed [Goodwin et al.,
1998; Hughes et al., 2013], to date, no information is available on bacterial production
of CH2Br2. In the open ocean mixed layer, CHClBr2 was also correlated with
heterotrophic bacteria. Moore [2003] indicated bacteria are unlikely to affect CHBr3
production, which supports our observations of a lack of correlation of CHBr3 with
bacteria. Our results suggest that heterotrophic bacteria may also contribute to
BrVSLS biogeochemistry in terms of their destruction and production.
In the open ocean water column, mean CHBr3 and CH2Br2 concentrations were
comparable in the mixed layer, below the mixed layer within the euphotic zone, and
below the euphotic zone. In contrast, CHBr3 concentrations in the coastal ocean mixed
layer were more than three times higher than CH2Br2. While these observations may
suggest differences in their formation and degradation rates in the coastal ocean and
open ocean, it is also possible that different DOM characteristics were influencing
such trends. Lin and Manley [2012] found that CH2Br2 was not always formed when
different DOM size fractions collected from various locations in California were
59
exposed to bromoperoxidase. This finding may indicate terrestrially derived DOM is
potentially more reactive for CHBr3 formation, compared to DOM derived in the
marine environment.
3.3.4 Correlations between CHBr3 and CH2Br2, CHClBr2, and CHBrCl2
Despite the differences in the degree of correlations, CHBr3 was significantly
correlated with CH2Br2 and CHClBr2 throughout the water column for the entire
HalocAST-P cruise (Table 3-3). Significant correlation between CHBr3 and CH2Br2
has been frequently interpreted as the two are derived from common sources.
However, CHBr3 and CH2Br2 significantly correlated with different biological
parameters during the HalocAST-P cruise. For example, heterotrophic bacteria
abundance only significantly correlated with CH2Br2 but not with CHBr3. Such a
pattern suggests different biogeochemical factors within the ecosystem can affect the
concentrations of these compounds separately. This finding is consistent with
observations by [Quack et al., 2007a] in the Mauritanian upwelling system. A recent
laboratory study by Hughes et al. [2013] found that CHBr3 and CH2Br2 concentrations
in seawater were controlled by different processes. The authors hypothesized that
CH2Br2 is transformed from CHBr3 via biologically mediated processes. Such a
hypothesis was supported by their experimental results and other lines of evidence
previously observed in the laboratory and field [Hughes et al., 2009; Tokarczyk and
Moore, 1994]. Therefore, the co-occurrence of CHBr3 and CH2Br2 is potentially due to
60
processes occurring in a common type of ecosystem and controlled by different
factors, rather than their being derived from a common biological source.
CHBr3 was only significantly correlated with CHBrCl2 in the mixed layer
(Table 3-3). It is interesting to consider CHBrCl2 below the euphotic zone, where
successive chlorine substitution of CHBr3 was proposed. CHBrCl2 was significantly
correlated with CHClBr2 (ρ = 0.55, p << 0.001, n =106) but not associated with CHBr3
below the euphotic zone, which may further support the idea of chlorine substitution,
as CHBrCl2 is not directly linked to CHBr3 in such a chemical process.
Table 3-3. Spearman’s rank correlation coefficient (ρ) of CHBr3 with CH2Br2, CHClBr2, and CHBrCl2, p-value and number of samples (n) are presented in the parentheses. “ns” indicates not significant correlation.
CH2Br2 CHClBr2 CHBrCl2 Mixed Layer
CHBr3
0.67 (<< 0.001; 78)
0.81 (<< 0.001; 78)
0.57 (<< 0.001; 78)
Below mixed layer within the euphotic zone
CHBr3 0.56
(<< 0.001; 35) 0.66
(<< 0.001; 35) 0.30ns
(0.08; 35) Below euphotic zone
CHBr3
0.70 (<< 0.001; 106)
0.29 (0.003; 106)
0.06ns (0.56; 106)
3.4 Conclusions
Our findings suggest BrVSLS production is in general related to
photosynthetic biomass production, and heterotrophic bacteria may be sources for
certain BrVSLS, for example, CH2Br2. However, the relationship between BrVSLS
production and photosynthetic biomass production is not straight forward, and likely
involves complex factors, such as phytoplankton species composition, interactions
61
between phytoplankton groups and/or bacterioplankton, and enzymatic reactions with
specific DOM moieties. Our results also suggest CHBr3 and CH2Br2 may be derived
from disparate sources and are controlled by difference biogeochemical processes in
certain regions. Finally, CHBrCl2 may have more significant sources in deeper waters
than biochemical sources found in the euphotic zone, and successive chlorine
substitution of CHBr3 may be one of them. To better understand source of the
BrVSLS and their feedbacks under climate change, fundamental studies concerning
their formation and degradation mechanisms are required. A better understanding of
these mechanisms is important as most of the anthropogenic ozone depleting
substances (ODS) were controlled by the Montreal Protocols, and the naturally
derived ODS will be more important in controlling ozone chemistry in the future
atmosphere.
62
CHAPTER IV
SPATIAL AND TEMPORAL DISTRIBUTIONS OF BROMOFORM AND
DIBROMOMETHANE IN THE ATLANTIC OCEAN AND THEIR
RELATIONSHIP WITH PHOTOSYNTHETIC BIOMASS
4.1 Introduction
Brominated very short-lived substances (BrVSLS), thought of as brominated
trace gases with atmospheric lifetimes of 0.5 year or less, are potentially significant
contributors to catalytic ozone loss in the troposphere and lower stratosphere [Montzka
and Reimann, 2011]. BrVSLS have received considerable attention because bromine
is more effective in depleting ozone than chlorine on a per atom basis [Garcia and
Solomon, 1994; Solomon et al., 1995]. Once emitted to the atmosphere, BrVSLS are
degraded either via reactions with hydroxyl radicals or photolysis in the atmosphere
resulting in inorganic bromine (Bry), which may then participate in catalytic ozone
destruction. Bromoform (CHBr3) and dibromomethane (CH2Br2) are the most
abundant BrVSLS accounting for ~ 80% of the very short-lived organic bromine in the
marine boundary layer [Law and Sturges, 2007]. Mean local atmospheric lifetimes for
CHBr3 and CH2Br2 are 24 days and 123 days, respectively [Montzka and Reimann,
2011]. Along with the minor VSLS constituents such as bromodichloromethane
(CHBrCl2), chlorodibromomethane (CHClBr2), and bromochloromethane (CH2BrCl),
these trace gases are capable of supplying 1 to 8 parts per trillion (ppt) of Bry to the
63
stratosphere, which is equivalent to ~ 4 to 36% of the total stratospheric bromine
[Montzka and Reimann, 2011].
CHBr3 and CH2Br2 are predominantly produced naturally in seawater and are
believed to be associated with phytoplankton and macroalgae [Carpenter and Liss,
2000; Goodwin et al., 1997; Manley et al., 1992; Moore et al., 1996; Sturges et al.,
1992; Sturges et al., 1993]. However, biological proxies, such as chlorophyll a,
usually do not significantly correlate with these BrVSLS. Currently, we do not have an
adequate conceptual model of the sources and formation mechanisms of the BrVSLS,
and the environmental forcing that influences BrVSLS production in the ocean. To
date, no prolific terrestrial natural sources for these two BrVSLS have been identified,
although small amounts of CHBr3 emissions were observed in a flooded rice paddy
[Redeker et al., 2003]. CHBr3 also has anthropogenic sources, as it is one of the by-
products of water disinfection [Quack and Wallace, 2003; Rook, 1974, 1976]. Despite
the significant amount of CHBr3 released from such a process, it only accounts for less
than 1 % of the total global emission [Quack and Wallace, 2003].
A number of recent studies reported CHBr3 and CH2Br2 sources, atmospheric
mixing ratios, seawater concentrations, and sea-to-air fluxes in different regions of the
world [Butler et al., 2007; Carpenter and Liss, 2000; Carpenter et al., 2009;
Carpenter et al., 2007b; Quack and Wallace, 2003; Quack et al., 2007a; Quack et al.,
2007b; Quack et al., 2004; Raimund et al., 2011]. However, large uncertainties remain
in estimating their global budgets because of the significant spatial and temporal
variations of CHBr3 and CH2Br2. This is especially true for CHBr3, which is not well-
mixed in the atmosphere owing to its short local lifetime [Montzka and Reimann,
64
2011]. Therefore, elevated CHBr3 atmospheric mixing ratios are usually confined to
their source regions. A model study showed that altering CHBr3 emission source
distribution scenarios from only the tropical open ocean to the tropical coastal and
open oceans combined would significantly alter the estimated annual global sea-to-air
flux from 5.0 to 7.4 Gmol Br yr-1 [Warwick et al., 2006]. While this finding highlights
the importance of the coastal ocean as a significant contributor to atmospheric CHBr3,
it also clearly shows that the global CHBr3 emission budget is sensitive to varying
source types and emission regions.
Quack and Wallace [2003] combined large amounts of data from different
studies conducted in different regions of the ocean to provide an estimate of the
CHBr3 global annual net sea-to-air flux. The authors noted, however, that different
measurement techniques and calibration methods used in the various studies may have
resulted in large discrepancies in their estimate. The authors also suggested that the
uncertainty in their global estimate largely depends on uncertainties in the saturation
anomalies. Moreover, extrapolation with relatively sparse and variable data, as well as
choice of different gas exchange coefficient parameterizations (kw) in different studies,
may also introduce large discrepancies in estimating global budget of CHBr3 and
CH2Br2. Finally, in the data compiled by Quack and Wallace [2003], atmospheric and
seawater measurements were not always paired, which highlighted the importance of
measuring atmospheric and seawater concentrations simultaneously in order to
minimize such uncertainties.
Here, we present air and seawater measurements of CHBr3 and CH2Br2 over a
large spatial extent and over a period of almost two decades. Importantly, CHBr3 and
65
CH2Br2 were measured using the same analytical technique during all of the cruises
discussed. Measurements of boundary layer air and of seawater were made
simultaneously and were calibrated against the National Oceanic and Atmospheric
Administration (NOAA) scale. The objective of this study is to employ datasets with
less variation in analytical and calibration techniques, which should reduce uncertainty
and improve our understanding of global distributions of CHBr3 and CH2Br2, their
marine sources, and their global annual net fluxes. BrVSLS data were collected from
five cruises from 1994 to 2010 (Figure 4-1; Table 4-1) in the Atlantic basin. A16 north
(A16N) and A16 south (A16S), which occurred in 2003 and 2005, are both part of the
Climate Variability and Predictability (CLIVAR) repeat hydrography project onboard
R/V Ronald H. Brown. The Halocarbon Air-Sea Transect – Atlantic (HalocAST-A)
cruise in 2010, onboard the FS Polarstern, was conducted in an attempt to repeat the
Bromine Latitudinal Air-Sea Transect II (BLAST-II) cruise conducted in 1994 [Butler
et al., 2007; Lobert et al., 1996], along similar latitudinal bands within a similar region
and season. BrVSLS data for the BLAST-II and Gas Exchange Experiment 98
(GaxEx98) cruises were obtained from the NOAA Earth System Research Laboratory
Global Monitoring Division database (http://www.esrl.noaa.gov/gmd/hats/ocean/)
[Butler et al., 2007; King et al., 2000; Lobert et al., 1996] and are also identified in the
HalOcAt database.
66
Figure 4-1. Ship tracks for the GasEx98 (red line), BLAST-II (grey line), A16N (magenta line), A16S (blue line), and HalocAST-A (green line) cruises.
67
Table 4-1. List of cruises presented in this study.
Expedition Start/End Points Season Research Vessel CLIVAR A16N
Leg B Reykjavik, Iceland (June 19, 2003) - Funchal, Madeira
(July 10, 2003)
Summer R/V Ron Brown (US)
Leg C Funchal, Maeria (July 15, 2003) - Natal, Brazil (August 10, 2003) Summer R/V Ron Brown (US)
CLIVAR A16S Leg 2 Punta Arenas, Chile (Jan 11, 2005) -
Fortaleza, Brazil (Feb 22, 2005) Fall R/V Ron Brown (US)
HalocAST-A
Bremerhaven, Germany (October 25, 2010) - Cape Town, South Africa (November 24, 2010)
Winter (NH) / Summer (SH) FS Polarstern (DE)
BLAST-II a,b Bremerhaven, Germany (October 18, 1994) - Punta Arenas, Chile (November 21, 1994)
Winter (NH) / Summer (SH) FS Polarstern (DE)
GasEx98b,c Leg 1 Miami, FL (May 7, 1998) - Lisbon, Portugal (May 25,
1998) Early summer (NH) R/V Ron Brown (US)
Legs 2 to 4 Lisbon, Portugal (June, 1998) - Miami, FL (July 15, 1998) Summer (NH) R/V Ron Brown (US)
References a. Lobert et al., 1996 b. Butler et al., 2006 c. King et al., 2000
4.2 Method
4.2.1 Gas Analyses
Trace gases were separated and analyzed using a cryo-trapping and focusing
system with gas chromatography-mass spectrometry (GC-MS) detection. Detailed
analytical system configurations were described in Yvon-Lewis et al. [2004].
Modifications made to improve trapping and drying performance over the course of
time are summarized in Butler et al. [2007]. For the HalocAST-A cruise, dry mole
fractions of the BrVSLS in the atmosphere and seawater were measured alternately
every ~52 minutes, or ~112 minutes when calibration standard was run in sequence,
which was after every forth injection. Dual calibration standards were used during the
HalocAST-A cruise to ensure measurement accuracy. Equilibrium partial pressures for
68
surface seawater were measured in headspace samples collected from a Weiss-type
equilibrator with continuous seawater flowing from the ship’s underway-pumping
system. Atmospheric samples were collected from a sampling line with its inlet
mounted on the mast at the bow of the ship. The samples were dried with two Nafion
dryers (Perma Pure, NJ) in series, cryo-trapped and focused at -80 °C, and then
desorbed at 200°C into a DB-VRX column (Agilent J&W; I.D. 0.18 mm; Film 1.0 µm;
10 m pre-column and 30 m main column). For the CLIVAR A16N and A16S cruises,
during which continuous underway measurements of the trace gases were not
available, discrete seawater samples were collected from CTD casts daily with 100 mL
glass syringes, and analyzed using an automated purge-and-trap system with a GC-MS
configured similar to the HalocAST-A cruise but with a different column (Agilent
J&W; I.D. 0.25 mm; Film 1.4 µm; 15 m pre-column and 45 m main column). Details
of the automated purge and trap system configuration are described in Yvon-Lewis et
al. [2004]. Purge efficiencies for CHBr3 and CH2Br2 determined for the two cruises
were > 82%. For the CLIVAR A16N and A16S cruises, air samples were collected
daily with stainless-steel canisters with the air inlet extended ~5 m above deck and
into the wind.
Calibration standards were whole air working-standards calibrated before and
after the cruises against a whole air working-standard obtained from the NOAA
Global Monitoring Division (GMD). Detection limits for CHBr3, CH2Br2, CHBrCl2
and CHClBr2 during these cruises were 0.02 ppt, 0.03 ppt, 0.01 ppt, and 0.01 ppt, or
lower. The largest analytical uncertainty was 7.0% for CHBr3, 6.0% for CH2Br2, and
9.0% for CHBrCl2 and CHClBr2. Analytical and calibration methods for the BLAST-II
69
and GasEx98 cruises were described in King et al. [2000] and Lobert et al. [1996] and
were similar to methods used in the later cruises.
All BrVSLS were measured in dry mole fraction. For atmospheric values, dry
mole fraction (atmospheric mixing ratio in ppt) was reported; hence, the values do not
vary with local humidity. For the equilibrated seawater values, the measured dry mole
fraction was corrected to partial pressure (in pico atmosphere, patm) and sea surface
conditions (temperature and seawater vapor pressure), and then converted to pico
molar (pmol L-1) using the gas constant and Henry’s Law constant (H) [Moore et al.,
1995a]. For seawater concentrations measured from discrete water samples (i.e. A16N
and A16S), concentrations were corrected to pmol L-1 with the known sample volume.
Atmospheric and seawater dry mole fractions were converted to patm using
appropriate water vapor corrections [Weiss and Price, 1980] for calculating saturation
anomalies (Δ) and fluxes (F).
4.2.2 Saturation anomaly and flux calculations
Saturation anomalies (Δ, %) are defined as percent deviation from equilibrium,
where positive Δ and negative Δ indicated the BrVSLS in seawater were
supersaturated and undersaturated relative to the atmosphere. Saturation anomalies
were calculated with the following equation:
Δ = pw - papa ×100% (Equation 4.1)
70
where, pw (patm) is the partial pressure for the surface seawater, corrected from partial
pressure measured in the equilibrator (peq, patm), and pa (patm) is the partial pressure
in the atmosphere.
The net sea-to-air fluxes (F, nmol m-2 d-1) for CHBr3 and CH2Br2 were
calculated with the following equation:
𝐹𝐹 = 𝑘𝑘w(Cw − !a!
) (Equation 4.2)
where, kw (m s-1) is the gas exchange coefficient using the Sweeney et al. [2007]
parameterization and corrected for the Schmidt number of the gases. Wind speeds
were corrected to 10 m (u10; m s-1) above the sea surface from the height where they
were originally measured [Erickson, 1993] for determining kw (Figure 4-2). The
Schmidt numbers for the BrVSLS were calculated from the kinematic viscosity of
seawater based on viscosity and density of the seawater [Millero, 1974; Millero and
Poisson, 1981] and diffusivities of the gases [Hayduk and Laudie, 1974]. H (L atm
mol-1) is the Henry’s law constant converted from the dimensionless Henry’s law
constant reported by Moore et al. [1995a]. Cw (mol L-1) is equilibrium concentrations
of the gas in seawater. pa (atm) is partial pressure of the gas in the atmosphere as
defined above.
71
Figure 4-2. 24 hours averaged wind speed at 10 m above sea surface (u10) for (a) the BLAST-II, A16N, A16S, and HalocAST-A cruises and (b) GasEx98 Legs 1, 2, 3, and 4.
72
4.2.3 Measurements of other biogeochemical variables
For the HalocAST-A cruise, a series of biogeochemical samples were collected
to better understand source distributions of CHBr3 and CH2Br2 and their relationships
with photosynthetic biomass in seawater. Each day, samples for picoplankton
abundance, dissolved nutrient concentrations, dissolved organic carbon (DOC)
concentration, and pigment concentrations were collected from the ship’s membrane
seawater-pumping system, which was designed for collecting underway
biogeochemical samples. To account for spatial change of ship’s location and
variability of biogeochemical and halocarbon data, all the biogeochemical
measurements were paired with the nearest halocarbon measurements based on
sampling location. While this is the most conservative approach to ensure less bias, it
is possible that different turnover times of each variable may lead to some
uncertainties in correlations between the BrVSLS and biogeochemical data.
Dissolved nutrients, urea (CH4N2O), nitrate (NO3-), nitrite (NO2
-), ammonium
(NH4+), phosphate (PO4
3-), and silicate (SiO44-), and DOC samples were filtered
through pre-combusted 0.7 µm nominal pore size GF/F filters and stored at -20 °C
until analysis. The dissolved nutrient samples were sent to the Geochemical and
Environmental Research Group (GERG) at Texas A & M University for analysis.
Dissolved nutrient samples were analyzed following well-established protocols on an
Astoria-Pacific Analyzer [Armstrong et al., 1967; Bernhardt and Wilhelms, 1967;
Harwood and Kühn, 1970]. DOC analyses were performed on a Shimadzu TOC-
73
VCSH/CSN analyzer using high-temperature catalytic oxidation (HTC), as described
by Guo et al. [1994].
For each pigment sample, 5 to 10 L of seawater were filtered through a pre-
combusted 0.7 µm nominal pore size GF/F filters until color were visible on the filters.
The filters were frozen immediately at -80 °C and kept frozen until analysis back in
the laboratory with high performance liquid chromatography (HPLC). The
phytoplankton pigment extraction procedure was followed as described in Wright et
al. [1991]. Details of the analytical method are described in [Liu et al., 2013],
following the analytical configuration described in Chen et al. [2003].
For flow cytometry sample collection, 1 mL of water collected from the flow-
through membrane pump was filtered into a round-bottom tube with a 35 µm mesh
cell strainer (BD Falcon) and fixed with 20 µL 10% paraformaldehyde for 10 minutes
(final concentration 0.2%). The fixed samples were then quickly frozen at -80 °C in
cryotubes and kept frozen until analysis. Picoplankton and heterotrophic bacteria were
counted with a Becton Dickinson FACSCalibur flow cytometer following
configuration and protocol described in Campbell [2001]. For non-pigmented,
heterotrophic bacteria, cells were stained with SYBR Green (Molecular Probes) prior
to introduction into the flow cytometer. Data were analyzed using CytoWIN [Vaulot,
1989].
74
4.2.4 Data handling and statistical methods
Exceptionally high or low values were re-verified for potential instrumental
error and removed if necessary. Abnormal values of BrVSLS concentrations due to
instrumental error were determined from CFC-11 within the same run as an inert
tracer. All data were tested for normality to determine the most appropriate statistical
method used for data interpretation. Spearman’s rank correlation was chosen to assess
the association between parameters, due to the non-normal distribution of the data.
Spearman’s rank correlation analyses were evaluated at the 95% confidence level.
Significance of correlation was determined using the two-tailed test. Significant
differences of means from different environmental regimes (see section 3) were
determined using the one-way analysis of variance (ANOVA). All statistical analyses
were done with the OriginPro© version 9.0 (OriginLab©) statistic module.
4.3 Results and discussion
To assess BrVSLS variability in different geographical regimes, the ocean was
divided into three regimes: open ocean, mid-ocean islands, and coastal ocean. The
mid-ocean island regime was defined as locations within 200 kilometers radius of any
mid-ocean islands. The coastal ocean regime was defined as locations with water
depth ≤ 200 m. To better assess the relationship between CHBr3 and CH2Br2 with the
presence of photosynthetic biomass, the open ocean regime was further divided into
three sub-regimes based on satellite observed chlorophyll a concentration: low
75
([chlorophyll a] < 0.1 mg m-3), medium (0.1 mg m-3 ≤ [chlorophyll a] < 0.2 mg m-3),
and high ([chlorophyll a] ≥ 0.2 mg m-3).
Upwelling regions, where nutrients are brought to the surface from deeper
waters, are unique ecosystems compared to the rest of the open ocean. Therefore,
during the HalocAST-A cruise, CHBr3 and CH2Br2 production in the upwelling
systems was examined using the biogeochemical data collected during this cruise. The
African upwelling zones were defined following Hurrell et al. [2006]. During the
HalocAST-A cruise, the ship encountered waters influenced by the Northwestern
African upwelling (7 - 25°N), equatorial upwelling (3°N - 5°S), and the Southwestern
African upwelling (10 – 27°S) systems. These definitions were supported by monthly-
time averaged primary production data, which were derived from chlorophyll a data at
a 9-km resolution retrieved from the Sea-viewing Wide Field-of-view Sensor
(SeaWiFS) during the time of the cruise [Acker and Leptoukh, 2007].
4.3.1 Temporal and spatial variations in CHBr3 and CH2Br2 concentrations
Data from the BLAST-II, GasEx98, A16N, A16S, and HalocAST-A cruises
show overall temporal variations of CHBr3 in its atmospheric mixing ratios and
seawater concentrations in the Atlantic basin (Figure 4-3). The A16N and the northern
hemispheric segment of BLAST-II cruises were conducted in similar regions but in
different seasons (Table 4-1). CHBr3 seawater concentrations and atmospheric mixing
ratios in this region were almost three times higher in the boreal summer (A16N)
compared to winter (BLAST-II), if we assume that inter-annual variations in CHBr3
76
production is less significant than seasonal variations (Figure 4-3, Table 4-2).
Although the differences for CH2Br2 were not as substantial, seawater concentrations
and atmospheric mixing ratios in this region were still higher during the boreal
summer (A16N). The HalocAST-A cruise data were not considered for this particular
comparison to avoid spatial effects, as the cruise track in the northern hemisphere is
closer to the continent than the A16N and BLAST II cruises (Figure 4-1).
Figure 4-3. Latitudinal distributions of CHBr3 and CH2Br2 atmospheric mixing ratios (a and c) and seawater concentrations (b and d) measured during the BLAST-II, A16N, A16S, and HalocAST-A cruises.
77
Table 4-2. Mean (range) of BrVSLS atmospheric mixing ratios, seawater concentrations, saturation anomalies, and fluxes during the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 in different hemispheres and seasons.
Atmospheric mixing ratio Seawater concentration Saturation anomaly Flux
(ppt) (pmol L-1) (%) (nmol m-2 d-1) Northern Hemisphere HalocAST-A (winter)
CHBr3 1.9
(0.3 to 3.9) 8.5
(0.08 to 38.0) 233.2
(-91.1 to 884.6) 12.1
(-3.8 to 71.8)
CH2Br2 1.0
(0.6 to 1.5) 2.3
(1.0 to 5.8) 133.2
(31.8 to 296.4) 3.0
(0.4 to 13.7)
CHClBr2 0.7
(0.5 to 1.2) 1.7
(0.9 to 2.4) 215.2
(64.2 to 501.2) 2.5
(0.3 to 7.6)
CHBrCl2 0.5
(0.3 to 0.8) 0.5
(0.3 to 2.6) 140.0
(-9.5 to 335.9) 0.4
(-1.2 to 2.0) GasEx98 Leg 1 (early summer)
CHBr3 0.5
(0.2 to 1.4) 1.5
(0.3 to 3.8) 57.9
(-51.6 to 213.2) 1.2
(-1.2 to 4.6)
CH2Br2 1.0
(0.3 to 2.0) 1.7
(0.5 to 3.3) 46.1
(-55.4 to 171.0) 1.2
(-1.8 to 6.5) GasEx98 Legs 2 to 4 (summer)
CHBr3 0.4
(0.1 to 4.6) 1.5
(0.1 to 5.5) 97.2
(-39.2 to 761.0) 1.7
(-2.9 to 13.6)
CH2Br2 1.3
(0.2 to 2.6) 2.8
(0.2 to 5.8) 83.5
(-21.1 to 490.2) 3.7
(-1.0 to 16.3) A16N (summer)
CHBr3 1.5
(0.7 to 5.9) 3.2
(0.9 to 6.4) 71.1
(-61.7 to 231.7) 3.5
(-8.4 to 12.1)
CH2Br2 1.3
(1.0 to 1.7) 2.0
(0.7 to 3.9) 48.0
(-40.8 to 116.4) 2.0
(-0.9 to 6.5) Blast II (winter)
CHBr3 0.6
(0.4 to 0.7) 1.1
(0.5 to 4.8) 30.2
(-10.6 to 369.4) 0.5
(-1.0 to 5.0)
CH2Br2 0.9
(0.6 to 1.3) 1.5
(0.6 to 8.0) 66.9
(-12.6 to 673.5) 1.7
(-1.3 to 9.7) Southern Hemisphere HalocAST-A (summer)
CHBr3 1.4
(0.1 to 2.2) 8.0
(1.5 to 12.7) 299.2
(57.9 to 2190.2) 14.1
(3.7 to 35.0)
CH2Br2 0.8
(0.7 to 0.9) 2.6
(1.8 to 3.4) 197.4
(72.6 to 382.2) 4.3
(1.6 to 7.8)
CHClBr2 0.6
(0.4 to 0.8) 2.0
(1.3 to 4.6) 308.2
(158.2 to 690.4) 3.6
(1.3 to 9.8)
CHBrCl2 0.3
(0.2 to 0.4) 0.6
(0.2 to 1.9) 383.3
(45.4 to 1272.1) 1.1
(0.3 to 4.3) A16S (Fall)
CHBr3 1.5
(0.1 to 4.3) 9.7
(0.3 to 73.8) 235.4
(13.8 to 1027.1) 8.4
(0.5 to 41.9)
CH2Br2 0.9
(0.3 to 1.4) 1.9
(0.3 to 10.6) 39.8
(-9.9 to 143.9) 0.9
(-1.1 to 5.6) Blast II (summer)
CHBr3 0.5
(0.2 to 0.8) 1.2
(0.5 to 3.4) 34.2
(-39.1 to 312.6) 0.9
(-1.5 to 7.6)
CH2Br2 0.8
(0.5 to 1.2) 1.6
(0.7 to 4.9) 66.2
(-15.9 to 307.5) 1.9
(-1.0 to 23.8)
78
In the southern hemisphere, the highest seawater concentrations and
atmospheric mixing ratios of CHBr3 were observed during the austral fall (A16S)
(Figure 4-3, Table 4-2). These differences may be influenced by seasonal variation in
CHBr3 production, and the fact that the A16S, HalocAST-A and BLAST-II cruises
were conducted in different regions of the southern Atlantic Ocean. Atmospheric
mixing ratios of CH2Br2 in the southern hemisphere were comparable among the
A16N, BLAST-II, and HalocAST-A cruises. Seawater concentrations of CH2Br2 were
comparable among the A16N and BLAST-II cruises between 0° to 40 °S (Figures 4-3c
and 4-3d). However, during the HalocAST-A cruises, seawater concentrations of
CH2Br2 were higher than those observed in the A16S and BLAST-II cruise in the
southern hemisphere (Figures 4-3c and 4-3d).
Data from all the cruises discussed in this study indicate that CHBr3 seawater
concentrations vary significantly across the Atlantic basin (Figures 4-3 and 4-4). To
eliminate seasonal effects, only HalocAST-A and BLAST-II data were compared,
since they were conducted in the same season. Seawater concentrations of CHBr3 were
substantially higher during the HalocAST-A cruise (Table 4-2), which was somewhat
east of BLAST-II. If we assume that inter-annual variations in CHBr3 production is
less significant than spatial variations, then the differences in seawater concentrations
between HalocAST-A and BLAST- II may have been due to the fact that the
HalocAST-A cruise was in closer proximity to the African continent and African
upwelling zones, but the BLAST-II cruise was further away from these systems. This
would also be true for the atmospheric mixing ratios for CHBr3. CH2Br2 seawater
concentrations measured during the HalocAST-A cruise were also higher than those
79
measured in the BLAST-II cruise, but the differences were not as substantial as CHBr3
(Table 4-2). CH2Br2 atmospheric mixing ratios were comparable during both cruises,
which is consistent with longer-lived gases being more homogeneously mixed in the
atmosphere than short-lived ones, like CHBr3.
Figure 4-4. Longitudinal distributions of CHBr3 and CH2Br2 atmospheric mixing ratios (a and c) and seawater concentrations (b and d) measured during the GasEx98 cruise Leg 1 and GasEx98 Legs 2 to 4.
The GasEx98 cruise data provide us with the opportunity to look into
longitudinal variations of CHBr3 and CH2Br2. This particular cruise had four segments
in the Atlantic Ocean, two of which were conducted over an almost identical cruise
track a month apart (i.e. Legs 1 and 3). This cruise provided us with a unique
80
opportunity to examine temporal variability of CHBr3 and CH2Br2 over a shorter time
scale in the same region. Significant spatial variability of CHBr3 and CH2Br2 was
observed during the GasEx98 cruise (Figure 4-4). CHBr3 and CH2Br2 seawater
concentrations and atmospheric mixing ratios were much higher during Leg 2, which
was conducted in an eddy system at higher latitude, and the segment of Leg 4 in the
Gulf of Mexico, than in the North Atlantic Gyre (i.e. Legs 1 and 3) (Figure 4-4 and 4-
5). Clear temporal variations for these two BrVSLS were observed. In the North
Atlantic gyre, CHBr3 and CH2Br2 seawater concentrations and atmospheric mixing
ratios were substantially higher during Leg 1, which took place in May (Figure 4-4),
than Leg 3, which took place in June and July.
CHBr3 and CH2Br2 seawater concentrations and atmospheric mixing ratios
presented in this study not only indicate strong temporal and spatial variations of these
BrVSLS in the Atlantic basin, they also provided a better picture of these BrVSLS on
a global scale. Butler et al. [2007] presented a comprehensive set of BrVSLS seawater
concentration and atmospheric mixing ratio data in the global ocean. The authors
mentioned the Pacific Ocean tends to have higher CHBr3 atmospheric mixing ratios
and seawater concentrations. Such conclusions, however, were based on datasets
heavily biased toward the Pacific Ocean, with the Atlantic Ocean data represented
only by the BLAST-II and GasEx98 cruises. In this study, the A16N, A16S, and
HalocAST-A cruises provided three additional datasets, which were measured in
different regions and seasons to provide a more complete picture in the Atlantic
Ocean. The results indicate that the Atlantic Ocean could have comparable or even
higher CHBr3 atmospheric mixing ratios and seawater concentrations than the Pacific
81
Ocean, which suggests source strengths are not significantly different between the two
ocean basins. These findings confirmed the observed, overall temporal and spatial
variations in BrVSLS in the Atlantic basin. Consequently, conclusions based on
limited amounts of data to determine the global BrVSLS distributions and budgets
should be treated with caution, as data from a single or only a few expedition(s) may
not be globally representative. Therefore, better data coverage both temporally and
spatially would be needed to better constrain the distributions and budgets of short-
lived compounds. Results observed from the GasEx98 cruise suggest that data with
higher temporal resolution are essential for better understanding of the dynamics of
CHBr3 and CH2Br2.
82
Figure 4-5. Data deviation from individual cruise mean of CHBr3 during the GasEx98 leg 1 (a), legs 2 to 4 (b), A16N (c), A16S (d), BLAST-II (e), and HalocAST-A (f) cruises, superimpose on 9-km resolution monthly averaged chlorophyll a concentration observed during the time of the cruise from SeaWiFS, except for the BLAST-II cruise, for which monthly climatology was used (NASA Giovanni [Acker and Leptoukh, 2007]). Time frame over which the data were acquired is labeled on each plot. Red data points indicate positive deviation and grey data points indicate negative deviation. Color bar indicates chlorophyll a concentration.
83
4.3.2 Saturation anomalies and fluxes
Results from these cruises show that CHBr3 and CH2Br2 were supersaturated in
most parts of the Atlantic basin, with only a few locations where CHBr3 and CH2Br2
were undersaturated in seawater (Figures 4-6 and 4-7). This observation is consistent
with findings reported elsewhere (e.g. Butler et al. [2007], Liu et al. [2011], and
Quack and Wallace [2003]). As there is no evidence supporting significant
biogeochemical removal of CHBr3, the observed undersaturations may be due to
physical effects, such as radiative cooling, mixing of water masses, temporal changes
in vertical circulation, or elevated atmospheric mixing ratios being occasionally
transported from elsewhere. Bacterial-mediated degradation of CH2Br2 has only been
studied in coastal water incubations and phytoplankton isolates [Goodwin et al., 1998;
Hughes et al., 2013]. Although conditions in these studies may not represent global
open ocean conditions, biological removal of CH2Br2 may still, in part, contribute to
the open ocean undersaturations observed in this study.
84
Figure 4-6. Latitudinal distributions of CHBr3 and CH2Br2 saturation anomalies (Δ) (a and c) and fluxes (b and d) calculated for the BLAST-II, A16N, A16S, and HalocAST-A cruises.
85
Figure 4-7. Longitudinal distributions of CHBr3 and CH2Br2 saturation anomalies (Δ) (a and c) and fluxes (b and d) calculated for the GasEx98 cruise Leg 1 and Legs 2 to 4.
Maximum fluxes (Figures 4-6 and 4-7) for CHBr3 and CH2Br2 were generally
noted in regions with high photosynthetic biomass, such as in high productivity open-
ocean waters and near shore (i.e. near mid-ocean islands and near the coasts) (Table 4-
3), presumably due to significant biological sources in these regions. These findings
are in agreement with other studies [Butler et al., 2007; Carpenter et al., 2009; Quack
and Wallace, 2003]. Most of the negative fluxes of CHBr3 and CH2Br2 were observed
in the temperate Atlantic Ocean, which is consistent with findings by Chuck et al.
[2005]. Fluxes measured over the open ocean were extrapolated to a global scale to
assess bromine contributions to the atmosphere. Assuming the CHBr3 and CH2Br2
86
open ocean fluxes measured in the Atlantic basin are globally representative, the mean
extrapolated global open ocean annual sea-to-air fluxes calculated from the five
cruises are 0.24 to 3.80 Gmol Br yr-1 and 0.11 to 0.77 Gmol Br yr-1 for CHBr3 and
CH2Br2, respectively (Table 4-4). To facilitate comparisons, the area of global open
ocean was defined following Quack and Wallace [2003], who extrapolated the
Atlantic open ocean flux to provide a global open ocean annual flux of 3 (-0.4 to 10)
Gmol Br yr-1. The mean extrapolated fluxes from the five cruises were within the
range estimated by Quack and Wallace [2003]. However, it should be noted that
global open ocean annual flux estimated by Quack and Wallace [2003] was calculated
using the Wanninkhof [1992] gas exchange coefficient (kw) parameterization. Using
the same parameterization to calculate fluxes for the five cruises resulted in an average
value that is about 46 % higher than using the Sweeney et al. [2007] parameterization
for CHBr3 and CH2Br2. While fully acknowledging the importance of coastal
contributions, extrapolation of global net fluxes including coastal fluxes was not
considered due to the scarceness of coastal data in this study (n = 6), which may not be
a legitimate representation of CHBr3 and CH2Br2 in coastal waters. Since coastal
environments differ significantly worldwide, extrapolating coastal data collected from
only a few coastal areas may not be appropriate for the BrVSLS [Liu et al., 2011].
87
Table 4-3. Mean (ranges) of BrVSLS atmospheric mixing ratios, seawater concentrations, saturation anomalies, and fluxes during the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises in different environmental regimes. Open ocean is defined as water depth > 200 m, mid-ocean island is defined as 200 km radius of mid-ocean islands, and coastal ocean is defined as water depth ≤ 200 m.
Atmospheric Mixing Ratio
(ppt) Seawater Concentration
(pmol L-1) Saturation Anomaly
(%) Flux
(nmol m-2 d-1) Open Ocean - Low Chlorophyll a ([chl a] < 0.1 mg m-3)
CHBr3 0.6
(0.09 to 5.9) 1.3
(0.1 to 16.9) 75.1
(-61.7 to 2190.2) 1.7
(-8.4 to 46.1)
CH2Br2 0.8
(0.2 to 2.0) 1.0
(0.2 to 4.4) 48.9
(-26.3 to 490.2) 1.0
(-0.5 to 5.5)
CHClBr2H 0.7
(0.5 to 1.1) 1.7
(1.3 to 2.4) 202.3
(68.2 to 331.4) 3.0
(1.2 to 5.4)
CHBrCl2H 0.4
(0.2 to 0.8) 0.5
(0.3 to 2.6) 142.0
(-9.5 to 415.1) 0.6
(-0.2 to 1.7)
Open Ocean - Medium Chlorophyll a (0.1 mg m-3 ≤ [chl a] < 0.2 mg m-3)
CHBr3 0.7
(0.1 to 2.5) 2.0
(0.1 to 11.4) 88.5
(-91.1 to 1027.1) 2.1
(-3.8 to 26.8)
CH2Br2 0.9
(0.2 to 2.0) 1.4
(0.3 to 5.0) 87.4
(-55.4 to 369.0) 1.8
(-1.8 to 9.9)
CHClBr2H 0.7
(0.5 to 1.2) 1.7
(0.9 to 2.6) 250.5
(82.6 to 408.0) 2.8
(0.3 to 7.0)
CHBrCl2H 0.4
(0.2 to 0.7) 0.4
(0.3 to 0.9) 197.9
(-8.9 to 500.5) 0.6
(-0.1 to 2.0)
Open Ocean - High Chlorophyll a ([chl a] ≥ 0.2 mg m-3)
CHBr3 0.6
(0.1 to 3.7) 2.9
(0.3 to 41.7) 84.5
(-51.6 to 578.4) 2.7
(-2.9 to 56.2)
CH2Br2 1.2
(0.5 to 2.5) 3.1
(1.2 to 5.8) 84.3
(-40.8 to 447.8) 3.5
(-1.3 to 23.8)
CHClBr2H 0.6
(0.4 to 0.9) 2.0
(1.3 to 4.6) 290.2
(64.2 to 690.4) 3.4
(0.7 to 9.8)
CHBrCl2H 0.3
(0.2 to 0.7) 0.7
(0.2 to 1.9) 380.3
(35.3 to 1272.1) 1.0
(-1.2 to 4.3)
Mid-ocean Islands
CHBr3 1.0
(0.1 to 4.3) 12.3
(0.4 to 73.8) 153.7
(-24.2 to 884.6) 8.3
(-0.2 to 71.8)
CH2Br2 1.2
(0.7 to 2.0) 3.4
(0.4 to 10.6) 171.4
(-9.5 to 673.5) 3.2
(-0.1 to 9.8)
CHClBr2H 0.6
(0.5 to 0.7) 2.36
(2.32 to 2.40) 415.2
(329.2 to 501.2) 3.78
(3.76 to 3.80)
CHBrCl2H 0.44
(0.42 to 0.45) 0.48
(0.45 to 0.51) 143.3
(134.0 to 152.5) 0.6
(0.5 to 0.7)
Coastal ocean
CHBr3 1.7
(0.5 to 4.6) 2.5
(1.5 to 4.2) 52.8
(22.2 to 107.6) 3.1
(0.4 to 7.3)
CH2Br2 1.5
(0.7 to 2.6) 2.3
(1.2 to 2.7) 15.3
(-0.2 to 68.8) 5.0
H: Data from the HalocAST-A cruise only.
88
Table 4-4. Extrapolated global open ocean net sea-to-air fluxes of CHBr3 and CH2Br2 (Gmol Br yr-1), based on data from the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises.
CHBr3 CH2Br2 HalocAST-A 3.80 0.75
GasEx98 Leg 1 0.40 0.25 GasEx98 Legs 2 to 4 0.54 0.77
A16N 1.09 0.41 A16S 1.71 0.11
BLAST-II 0.24 0.38
4.3.3 CHBr3 and CH2Br2 concentrations in different environments and their
relationship with ocean photosynthetic biomass
CHBr3 and CH2Br2 are predominantly produced by natural sources in the
marine environment, and their production may be partly attributable to enzyme-
mediated halogenation of organic matter (OM) by hypobromous acid (HOBr). HOBr
is formed via haloperxoidase-mediated oxidation of bromine in seawater, in the
presence of hydrogen peroxide (H2O2) [Theiler et al., 1978; Wever et al., 1993].
Subsequently, HOBr can react with a wide range of OM in the surrounding waters and
formed halogenated organic molecules, including the volatile gases such as CHBr3
[Theiler et al., 1978; Wever et al., 1993]. Two haloperoxidases, bromoperoxidase and
iodoperoxidase, are found in some phytoplankton (such as diatoms and cyanobacteria)
and in various macroalgae [Hill and Manley, 2009; Johnson et al., 2011; Moore et al.,
1996; Wever et al., 1993]. Laboratory studies have identified BrVSLS production
from certain specific taxa of diatoms and coccolithophores [Colomb et al., 2008;
Moore et al., 1996].
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Numerous efforts have been made to understand the relationship between
CHBr3 production and photosynthetic biomass [Abrahamsson et al., 2004; Karlsson et
al., 2008; Liu et al., 2013; Mattson et al., 2012; Quack et al., 2007a; Raimund et al.,
2011; Roy, 2010]. Schall et al. [1997] and Carpenter et al. [2009] found that CHBr3
seawater concentrations are usually elevated in regions with high chlorophyll a
concentrations. However, correlations between CHBr3 seawater concentrations and
chlorophyll a concentrations are often not straightforward. Several studies have
showed that correlations between CHBr3 seawater concentrations and chlorophyll a
concentrations are statistically insignificant [Abrahamsson et al., 2004; Carpenter et
al., 2009; Hughes et al., 2009; Liu et al., 2011; Mattson et al., 2012]; yet, another
study observed significant positive correlations between the two in certain water
masses in the Mauritanian upwelling system [Quack et al., 2007a]. These studies
suggested that sea-to-air fluxes of CHBr3, mixing in seawater, different production
rates among the phytoplankton species, and degradation in seawater may have altered
correlations between CHBr3 concentrations and chlorophyll a concentrations. In the
urbanized coastal ocean, it is also possible that the correlation between CHBr3 and
chlorophyll a is masked in part by anthropogenic sources of CHBr3, such as the water
disinfection processes [Liu et al., 2011]. The authors also suggest that
photodegradation of CHBr3 could be significant in shallow coastal waters, which may
have also altered the correlation between CHBr3 and chlorophyll a.
Ocean color observed from satellites facilitates looking for trends on a large
spatial scale. As discussed above, various factors can alter the correlation between
CHBr3 seawater concentrations and chlorophyll a concentrations. Therefore, we used
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an alternate approach to assess the relationship between CHBr3 production and
chlorophyll a concentrations. Deviation from individual cruise mean (10th to 90th
percentile) was used to relate elevated CHBr3 seawater concentrations with satellite-
observed chlorophyll a concentrations. A positive deviation indicates elevated CHBr3
seawater concentrations and a negative deviation indicates CHBr3 seawater
concentrations below the cruise means. Time-averaged 9-km resolution SeaWiFS
ocean color data were downloaded from NASA Giovanni [Acker and Leptoukh, 2007].
For the GasEx 98, A16N, A16S, and HalocAST-A cruises, monthly time-averaged
data during the time of the cruises were used. For the BLAST-II cruise, when satellite
data during the expedition was not available, climatology derived from data from1997
to 2011, October to November, monthly-averaged data was used as our best estimate.
On a basin-wide scale, most of the positive deviations were located in or near regions
with higher chlorophyll a concentrations (Figure 4-5), which were consistent with
observations by Carpenter et al. [2009] in the tropical and North Atlantic Ocean.
To better assess CHBr3 and CH2Br2 distributions in different environments
with different ecosystems and their concentration gradients as a function of
photosynthetic biomass abundance, data from all five cruises were combined and
binned into environmental regimes as described at the beginning of section 4.3. Low,
medium, and high chlorophyll a regimes represent different ecosystems in the open
ocean. The mid-ocean island regime represents ecosystems where biogeochemical
constituents in the surrounding seawater may have been influenced by the islands and
coastal upwelling systems. Based on satellite retrieved chlorophyll a concentrations
and pigment analysis for the HalocAST-A cruise, waters near the mid-ocean islands
91
tend to have relatively low photosynthetic biomass compared to mainland coastal
waters and upwelling regions.
Figure 4-8. Boxplots of data from the A16N, A16S, HalocAST-A, BLAST-II and GasEx98 cruises in different geographical regimes (a and c). Due to the scarcity of coastal data from these cruises (n = 6), coastal data collected from the Gulf of Mexico and East Coast Carbon (GOMECC) cruise [Liu et al., 2011] were added to the coastal data when creating the boxplot and serve as a reference for representative comparison (grey shaded box). The open ocean regime is further divided into regimes with low ([chl a] < 0.1), medium (0.1 ≤ [chl a] < 0.2), and high ([chl a] ≥ 0.2) chlorophyll a concentrations (b and d). Line in the box indicates median of data, filled square indicates mean of data, box range indicates 25th to 75th percentile of data, whisker indicates 10th to 90th percentile of data, open box indicates 5th to 95th percentile of data. Stars indicate minimum and maximum of data.
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In the Atlantic basin, there were distinct CHBr3 and CH2Br2 seawater
concentration gradients ranging from high to low in waters near the mid-ocean islands,
and open ocean, respectively (Figures 4-8a and 4-8c). In the open ocean, CHBr3 and
CH2Br2 also exhibited seawater concentration gradients ranging from high to low in
high, medium, and low chlorophyll a concentration waters (Figures 4-8b and d), which
was consistent with the distributions of their concentration deviations from the cruise
means (Figure 4-5).
Results from this study suggest that CHBr3 and CH2Br2 concentrations are
qualitatively related to ocean photosynthetic biomass and/or any biogeochemical
processes that are associated with photosynthetic biomass. This relationship was most
evident during the GasEx98 cruise, during which Legs 1 and 3 passed through the
same open ocean region at different times. During Leg 1 of the GasEx98 cruise, the
ship was sailing near a “chlorophyll front” and positive values for deviations from the
cruise mean were observed along the transit. However, during Leg 3, the retreating
chlorophyll front in the temperate region led to the majority of the data observed in the
North Atlantic gyre exhibiting negative deviations (Figure 4-5). In fact, as shown in
section 4.3.1, CHBr3 and CH2Br2 seawater concentrations and atmospheric mixing
ratios were substantially higher during Leg 1, which may be explained by the different
phytoplankton abundance, as reflected in differences in chlorophyll a concentrations
between the two legs.
The temporal and spatial differences in CHBr3 concentrations discussed in
section 4.3.1 may also be explained by variations in chlorophyll a concentrations. In
addition to seasonal differences, CHBr3 seawater concentrations measured during the
93
HalocAST-A cruise were generally higher than the other cruises (Figures 4-3 and 4-4).
This may be attributed to influences of African upwelling waters encountered during
the HalocAST-A cruise, where primary production is, in general, higher than the rest
of the open ocean (Figure 4-5). In the northern hemisphere, CHBr3 seawater
concentrations were higher in the boreal summer (A16N) than early winter (BLAST-
II). This was likely a function of the relative intensities of the phytoplankton bloom
occurred in the equatorial upwelling zone during each cruise (Figure 4-5). It has been
shown that the equatorial phytoplankton bloom is usually more intense during the
boreal summer than during early winter [Grodsky et al., 2008]. In the southern
hemisphere, substantially elevated CHBr3 and CH2Br2 were also observed in the high
chlorophyll a regions during the austral fall (A16S). CHBr3 and CH2Br2
concentrations were lower and less variable in oligotrophic waters during austral fall
(Figures 4-3 and 4-5).
While our results suggest using satellite observed chlorophyll a concentrations
to relate BrVSLS concentrations and emissions in model simulations seem to be a
legitimate approach, using such an approach is still a rather crude and qualitative
approach. Spearman’s rank correlation (ρ) between satellite observed chlorophyll a
and shipboard measured CHBr3 concentrations varied from cruise to cruise, which
ranged from 0.07 to 0.79 (Figure 4-9a, Table 4-5), which confirmed that the
relationship between CHBr3 and chlorophyll a is not always straightforward.
Furthermore, CHBr3 water concentrations varied significantly within each chlorophyll
a concentration gradient, which suggested CHBr3 production strength varied greatly
among areas (Figures 4-8a and b). Such variations may be explained by the complex
94
factors that control bromoperoxidase-mediated reactions, such as species-specific
production of bromoperoxidase [Hill and Manley, 2009], specific moieties of DOC
that are susceptible to halogenation and release CHBr3 [Lin and Manley, 2012],
availability of H2O2 [Abrahamsson et al., 2003], and organismal interactions within
the ecosystem. Therefore, photosynthetic biomass abundance alone is not sufficient to
resolve the complex dynamic of CHBr3 in seawater. Hence, further investigation of
other parameterizations to constrain CHBr3 emission in model studies should be
considered. In the Atlantic Ocean, chlorophyll a and CH2Br2 were generally correlated
significantly, and their relationships did not vary as substantially as CHBr3 (Figure 4-
9b). Spearman’s rank correlations (ρ) between chlorophyll a and CH2Br2 calculated
for the five cruises ranged from 0.32 to 0.81 (Table 4-5). CH2Br2 seawater
concentration variability in each open ocean regime was not as substantial as CHBr3
(Figure 4-8d). Based on the results in this study, it is reasonable to assume that
CH2Br2 may have a more direct relationship with chlorophyll a concentrations than
CHBr3, in the open ocean (Table 4-5).
95
Figure 4-9. Scatter plots of CHBr3 (a) and CH2Br2 (b) concentrations vs. chlorophyll a concentration, for the BLAST-II, GasEx98, A16N, A16S, and HalocAST-A cruises.
96
Table 4-5. Spearman’s rank correlation coefficient (ρ) between chlorophyll a concentrations with CHBr3 and CH2Br2 open ocean seawater concentrations, based on data from the A16N, A16S, HalocAST-A, BLAST-II, and GasEx98 cruises, and all data combined. p-value and number of samples (n) are in parentheses. “ns” indicates the correlation is not significant. Note that unless it is indicated in the table, correlation coefficient is based on satellite observed chlorophyll a data.
CHBr3 CH2Br2
HalocAST-A 0.37 (<< 0.001; 102)
0.53 (<< 0.001; 101)
HalocAST-A (HPLC) 0.27nc (0.29; 51)
0.63 (0.008; 49)
GasEx98 Leg 1 0.07nc (0.49; 83)
0.59 (<< 0.001; 101)
GasEx98 Legs 2 to 4 0.33 (<< 0.001; 501)
0.65 (<< 0.001; 512)
A16N 0.46 (0.004; 38)
0.32 (0.05; 37)
A16S 0.79 (<< 0.001; 49)
0.81 (<< 0.001; 49)
BLAST-II 0.76 (<< 0.001; 430)
0.79 (<< 0.001; 433)
All data 0.47 (<< 0.001; 1214)
0.77 (<< 0.001; 1230)
4.3.4 A closer look into CHBr3 and CH2Br2 sources during the HalocAST-A
cruise
While the ocean color observed from satellite provided valuable insights to the
relationship between BrVSLS concentration and photosynthetic biomass abundance, it
ignores a considerable amount of detail needed to better understand BrVSLS sources
in the ocean. During the HalocAST-A cruise, samples for pigments, nutrients, and
DOC concentrations, as well as picoplankton and heterotrophic bacteria abundances
were collected, to examine the relationship between marine biogeochemical processes
and BrVSLS production in seawater. Several recent studies have utilized pigment
biomarker information to identify possible phytoplankton groups that may contribute
to CHBr3 production in seawater [Abrahamsson et al., 2004; Karlsson et al., 2008; Liu
97
et al., 2013; Mattson et al., 2012; Quack et al., 2007a; Raimund et al., 2011; Roy,
2010]. Some of these studies identified phytoplankton groups such as cyanobacteria,
diatoms, and prymnesiophytes as potential CHBr3 and CH2Br2 producers.
It is essential to gain more detailed information on phytoplankton in situ
composition to better understand BrVSLS sources given the fact that their production
appears to be species specific (e.g. Colomb et al. [2008], Hill and Manley [2009],
Hughes et al. [2011], Moore et al. [1996], and Tokarczyk and Moore [1994]).
Phytoplankton community composition based on pigments is not sufficient to attribute
BrVSLS production to specific species or genera. For example, zeaxanthin is
commonly used as a pigment biomarker for cyanobacteria [Bianchi and Canuel,
2011]; but the two most important cyanobacterial genera in the ocean (Synechococcus
and Prochlorococcus) cannot be distinguished by zeaxanthin alone. These two genera
can easily be distinguished and enumerated using flow cytometry, based on their sizes
and fluorescence properties [Campbell, 2001]. Moreover, flow cytometry provided
valuable information on picoeukaryote (< 3 µm algae including chlorophytes,
pelagophytes, and haptophytes) counts and heterotrophic bacteria counts.
During the HalocAST-A cruise, seawater CHBr3 concentrations were
significantly correlated with Synechococcus and picoeukaryote abundances (Table 4-
6). However, CHBr3 seawater concentrations were not significantly correlated with
any of the phytoplankton pigment biomarkers, which is consistent with Abrahamsson
et al. [2004] and Mattson et al. [2012]. Although our results agree with the two prior
studies in acknowledging pigment biomarkers are probably not reliable for identifying
CHBr3 producers, we suggest a more detailed analysis of phytoplankton composition
98
may provide better insights into CHBr3 sources. Our results are consistent with
Karlsson et al. [2008] and Quack et al. [2007a], who identified cyanobacteria as
potential sources for CHBr3. The insignificant correlation with cyanobacteria
biomarker zeaxanthin is likely due to the presence of Prochlorococcus, which
significantly contribute to zeaxanthin concentrations but were probably not
responsible for CHBr3 production in our study region, as the correlations between
Prochlorococcus abundances and BrVSLS concentrations were not significant (Table
4-6).
During the HalocAST-A cruise, CH2Br2 seawater concentrations were
significantly correlated with Synechoccocus, picoeukaryotes, total chlorophyll a, and a
series of phytoplankton pigments that could indicate contributions from diatoms and
prymnesiophytes (Table 4-6). CH2Br2 seawater concentrations were also significantly
correlated with heterotrophic bacteria, which is consistent with findings from the
Halocarbon Air-Sea Transect – Pacific (HalocAST-P) cruise [Liu et al., 2013].
However, there are no studies that have demonstrated the bacterial production of
CH2Br2 in a way that can unequivocally be used to determine their significance in
regulating surface water concentrations in the ocean. Heterotrophic bacteria
abundance is generally positively correlated with chlorophyll a concentrations,
making it difficult to separate the role of heterotrophic bacteria and phytoplankton in
CH2Br2 dynamics.
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Table 4-6. Spearman’s rank correlation coefficient (ρ) between biological variables with CHBr3 and CH2Br2 open ocean seawater concentrations during the HalocAST-A cruise, p-value and number of samples (n) are in parentheses. “ns” indicates the correlation is not significant. Only parameters exhibit significant correlation (p < 0.05) are shown.
Proc Syn picoeuk Hbac Tchl a Chl c2 Chl c3 Fuco 19'-but 19'-hex Diad Open ocean - Entire cruise CHBr3 ns 0.33
(0.03; 51) 0.33
(0.03; 51) ns ns ns ns ns ns ns ns
CH2Br2 ns 0.38 (0.01; 51)
0.63 (<< 0.001; 51)
0.46 (<< 0.001; 58)
0.57 (0.01; 23)
0.78 (<< 0.001; 23)
0.75 (<< 0.001; 23)
0.80 (<< 0.001; 23)
0.78 (<< 0.001; 23)
0.76 (<<0.001; 23)
0.77 (<<0.001; 23)
Open ocean - Northwestern African upwelling influenced
CHBr3 ns 0.70 (0.03; 9) ns ns ns ns ns ns ns ns ns
CH2Br2 ns ns ns ns ns ns ns ns ns ns ns
Open ocean - Equatorial upwelling CHBr3 ns ns ns ns ns ns ns ns ns ns ns
CH2Br2 ns ns 0.90 (0.04; 6) ns ns ns ns ns ns ns ns
Open ocean - Southwestern African upwelling influenced CHBr3 ns ns ns ns ns ns ns ns ns ns ns
CH2Br2 -0.54
(0.01; 20) 0.54
(0.04; 15) 0.55
(0.03; 15) 0.69
(0.002; 18) ns ns ns 0.89 (0.007; 7) ns ns 0.79
(0.04; 7) Abbreviations: Proc (Prochlorococcus), Syn (Synechococcus), Picoeuk (Picoeukaryotes), Hbac (Heterotrophic bacteria), Tchl a (total chlorophyll a), Chl c2 (chlorophyll c2), Chl c3 (chlorophyll c3), Fuco (fucoxanthin), 19'-but (19’-butanoyloxyfucoxanthin), 19'-hex (19’-hexanoyloxyfucoxanthin), Diad (diadinoxanthin).
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CHBr3 and CH2Br2 seawater concentrations correlate with different
biogeochemical variables in different upwelling systems (Table 4-6). While CHBr3
and CH2Br2 are thought to be derived from common sources, results from this study
and the HalocAST-P cruise in the Pacific Ocean [Liu et al., 2013] suggests that there
are also processes which affect the production of each gas separately. Similar
relationships were also observed in the Mauritanian upwelling system by Quack et al.
[2007a], who observed CHBr3 and CH2Br2 correlated with different pigment
biomarkers.
In waters off the Canary Islands (~28 °N), significant increases in CHBr3 and
CH2Br2 seawater concentrations were observed (Figure 4-3). The maxima of CHBr3
and CH2Br2 during the HalocAST-A cruise were also observed in this region, and
corresponded to low chlorophyll a concentrations. These findings were consistent with
Carpenter et al. [2009] observations in the same area. While such a feature contradicts
with the general trend between the BrVSLS and chlorophyll a concentrations observed
in this and other studies, it also highlights the importance of mid-ocean island
environments as unique ecosystems that may contribute considerable amounts of
BrVSLS to the adjacent atmosphere. Urea concentrations and heterotrophic bacteria
abundances were also elevated in this area. Urea has been used as an indicator of
bacterial degradation of amino acids [Remsen, 1971; Zehr and Ward, 2002]. While
there was no direct evidence that bacterial cycling of amino acids related to CHBr3
and CH2Br2 production, it may suggest unique DOC sources around the mid-ocean
islands, which may be more reactive for the enzyme-medicated formation of these
BrVSLS. Although it is difficult to assess potential anthropogenic influences on the
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BrVSLS concentrations in this region, such a sources cannot be excluded, as water
treatments in coastal waters may be significant sources for CHBr3 as well [Liu et al.,
2011; Quack and Wallace, 2003].
DOC concentrations were measured during the HalocAST-A cruise in order to
estimate the impact of DOC concentrations on CHBr3 and CH2Br2 concentrations.
However, DOC concentrations were not significantly correlated with CHBr3 and
CH2Br2 seawater concentrations, which may suggest that specific moieties within the
bulk DOC pool are required as organic substrates for enzyme-mediated halogenation.
For example, Theiler et al. [1978] found that ketones are better organic substrates than
phenols for a bromoperoxidase extracted from red algae. Lin and Manley [2012] also
found that CHBr3 and CH2Br2 production rates were functions of DOC quality and
varied significantly depending on the DOC origins, which suggest that bulk DOC
concentration was not an effective indicator of BrVSLS production.
During the HalocAST-A cruise, seawater concentrations of CHBr3 and CH2Br2
were significantly correlated (ρ = 0.53, p << 0.001, n = 101), but the correlations were
lower than those observed in other studies [Carpenter and Liss, 2000; Carpenter et al.,
2009; Liu et al., 2011; Quack et al., 2007a]. This observation reflects the fact that
these gases likely have different sources and were influenced by different
biogeochemical processes in different regions. While CHClBr2 and CHBrCl2 were not
specifically discussed in this study, it is worth mentioning that they only weakly
correlated with CHBr3 (ρ < 0.40, p < 0.01, n > 100).
It should also be noted that although CH2Br2 and CHBr3 seawater
concentrations correlated significantly in most of the cruises, their relationships varied
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from cruise to cruise (Figure 4-10). Significant correlations between CHBr3 and
CH2Br2 seawater concentrations were observed during the BLAST-II (ρ = 0.86, p <<
0.001, n = 450), A16N (ρ = 0.79, p << 0.001, n = 37), A16S (ρ = 0.98, p << 0.001, n =
54), and GasEx98 Legs 2 to 4 (ρ = 0.60, p << 0.001, n = 516) cruises. However,
CHBr3 and CH2Br2 were not significantly correlated with each other during GasEx98
cruise Leg 1 (ρ = 0.17, p = 0.12, n = 89). These results confirmed findings deduced
from the HalocAST-A cruise and suggested that CHBr3 and CH2Br2 formation may
have, in part, been due to different biogeochemical processes. Different DOC
chemical properties in different waters may also affect the ratio between CHBr3 and
CH2Br2 concentrations, which ultimately will influence the correlation between the
two. Lin and Manley [2012] found that CH2Br2 was not always formed when treating
different DOC size fraction with vanadium bromoperoxidase. Results presented by Lin
and Manley [2012] indirectly confirmed by findings of Hughes et al. [2013], whose
experimental results suggest CH2B2 is biologically transformed from CHBr3, rather
than from the same enzyme-mediated reaction that yields CHBr3.
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Figure 4-10. Scatter plots of CHBr3 seawater concentration vs. CH2Br2 seawater concentration for the BLAST-II, GasEx98, A16N, A16S, and HalocAST-A cruises.
4.4 Conclusion
Given the heterogeneity of CHBr3 in the ocean, estimated global CHBr3
budgets may be biased if the data used is not diverse enough in space and time.
Although the A16N, A16S, and HalocAST-A cruises add substantial amount of data to
the record, more data are needed to better estimate the global budget of these and other
trace gases with short lifetimes. Our results further suggest that CHBr3 and CH2Br2
production may be attributable to biogeochemical processes related to photosynthetic
biomass, such as interactions with heterotrophic bacteria, grazing, and DOC cycling.
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However, chlorophyll a and pigment biomarkers alone are not sufficient to explain
CHBr3 and CH2Br2 concentrations and production in seawater. CHBr3 and CH2Br2
seawater concentrations and production are influenced by different biogeochemical
processes, and these trace gases may potentially be derived from disparate sources in
certain regions. The complexity within the ecosystem and its impact on the BrVSLS
concentrations and production suggests that additional information on the
biogeochemical drivers of these gases are needed to better understand the sensitivity
of their distributions and fluxes in relation to climate change. This is especially
important, as many of the anthropogenic, ozone depleting substances (ODS)
controlled by the Montreal Protocol continue to decrease in abundance, ultimately
leaving naturally produced ODS as the source of ozone-depleting substances in the
atmosphere.
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CHAPTER V
MARINE DISSOLVED ORGANIC MATTER (DOM) COMPOSITION
DRIVES THE PRODUCTION AND CHEMICAL SPECIATION OF
BROMINATED VERY SHORT-LIVED SUBSTANCES
5.1 Introduction
Bromoform (CHBr3), dibromomethane (CH2Br2), chlorodibromomethane
(CHClBr2), and bromodichloromethane (CHBrCl2) constitute a significant fraction of
atmospheric brominated very short-lived substances (BrVSLS), which defined as
brominated trace gases with atmospheric lifetimes < 0.5 year. The major very short-
lived substances (VSLS), CHBr3 and CH2Br2, account for ~ 80% of the very short-
lived organic bromine in the marine boundary layer [Law and Sturges, 2007]. Though
only present at trace concentrations, BrVSLS play an important role in catalytic ozone
destruction cycles, as bromine is more efficient in destroying ozone than chlorine, on
an atom-by-atom basis [Garcia and Solomon, 1994; Solomon et al., 1995]. CHBr3,
CH2Br2, CHClBr2, and CHClBr2 can contribute a significant amount of inorganic
bromine (Bry) to the atmosphere via photolysis and reaction with the hydroxyl radicals
(OH). The resulting Bry can then participate in catalytic ozone destruction in the
troposphere and stratosphere. CHBr3, CH2Br2, CHClBr2, CHBrCl2, and other VSLS
constituents combined are capable of supplying 1 to 8 parts per trillion (ppt) of Bry to
the stratosphere (BryVSLS), which is equivalent to ~ 4 to 36% of the total stratospheric
106
bromine [Montzka and Reimann, 2011]; with naturally emitted CHBr3 and CH2Br2
account for a large fraction of the BryVSLS [Hossaini et al., 2012a].
CHBr3, CH2Br2, CHClBr2, and CHBrCl2 are mainly derived from natural
sources in seawater. As most of the anthropogenic ozone depleting substances (ODSs),
such as methyl bromide (CH3Br) and halons, have phased out under the Montreal
Protocol, the naturally derived ODSs are becoming more important in controlling
ozone chemistry. These natural BrVSLS are sensitive to climate change in multiple
aspects. First, it was predicted that stronger tropical conventions in response to future
climate change could transport more CHBr3 to the stratosphere [Hossaini et al.,
2012b]; while alteration in OH demands in the atmosphere in a future climate also
favor source gas injections (SGI) of CH2Br2, CHClBr2, and CHBrCl2 [Hossaini et al.,
2012b]. Second, these naturally produced BrVSLS are thought to be associated with
macro- and micro-algal production by an enzyme-mediated pathway. It has been
suggested that haloperoxidases, such as the vanadium bromoperoxidases (V-BrPO),
catalyze oxidation of halogens in the presence of hydrogen peroxide (H2O2) and form
reactive halogen intermediates, such as hypobromous acid (HOBrenz). The resulting
HOBrenz can halogenate a wide range of organic matter (OM), and subsequently
release halogenated trace gases, such as CHBr3 [Butler and Carter-Franklin, 2004; Lin
and Manley, 2012; Manley and Barbero, 2001; Moore et al., 1996; Theiler et al.,
1978; Wever and Van der Horst, 2013; Wever et al., 1993]. The components required
for CHBr3 formation under the enzyme-mediated pathway will likely influence by
climate change. Therefore, it is important to understand the production and controlling
107
factors of BrVSLS, which are the key for assessing their global emissions,
distributions, and responses to future climate change.
For more than a decade, scientists devoted to understand CHBr3, CH2Br2,
CHClBr2, and CHBrCl2 natural sources have used various analytical tools to
understand their source types and the environmental forcing affecting the production
of these compounds. Most researchers have hypothesized that there is a direct
relationship between BrVSLS production and the composition and biomass of the
phytoplankton community. This approach assumes that our lack of understanding of
BrVSLS production in situ stems from a failure to identify the phytoplankton taxa
responsible. Chlorophyll a and other accessory pigments have been widely used to
understand contributions from specific taxa of photosynthetic biomass to the
production of CHBr3, CH2Br2, CHClBr2, and CHBrCl2 [Abrahamsson et al., 2004;
Ekdahl et al., 1998; Karlsson et al., 2008; Liu et al., 2013; Liu et al., Submitted;
Mattson et al., 2012; Quack et al., 2007a; Roy et al., 2011]. However, it had been
shown that the relationships between the phytoplankton biomass proxies and BrVSLS
are not straightforward [Abrahamsson et al., 2004; Liu et al., 2013; Liu et al.,
Submitted; Mattson et al., 2012]. Hence, pigment biomarkers may not be a useful
indicator for BrVSLS production and there may not be a direct relationship between
phytoplankton community composition or biomass and BrVSLS production.
Manley and Barbero [2001] demonstrated that the removal of DOM from
natural seawater reduced CHBr3 production, which highlighted the essential role of
DOM in controlling production of CHBr3 and other trace gases produced under the
same pathway. More recently, Lin and Manley [2012] confirmed the important role of
108
dissolved organic matter (DOM) in controlling CHBr3 production in natural seawater,
which had been hypothesized for decades [Theiler et al., 1978; Wever and Van der
Horst, 2013; Wever et al., 1993]. The authors found a significant correlation between
DOM molecular size and CHBr3 production in V-BrPO treated natural seawater
collected from the California coast. Evidence presented by Lin and Manley [2012]
suggested the importance of DOM type and reactivity to halogenation and BrVSLS
production, which has important implications for the global BrVSLS biogeochemical
cycle. In this study, I examine the role of DOM in BrVSLS production in greater
detail.
5.2 Methods
5.2.1 Preparation of DOM model compound stocks
A total of 28 model DOM compounds were examined for their ability to form
BrVSLS upon halogenation by V-BrPO induced HOBrenz (Table 5-1). All DOM
model compounds were purchased from Sigma-Aldrich® at their highest purity grade.
To minimize DOM contamination during sample preparation, all the glassware used
was combusted at 500°C for 6 hours. To avoid significant change in seawater
properties, all compounds were dissolved in 0.2 µm filtered ultra-pure water (UHPW).
Sub-boiling UHPW was used to dissolve tyrosine, ferulic acid, syringaldehyde, starch,
and humic acid. Due to difficulty in completely dissolving humic acid, which
normally dissolves in basic solutions, the bulk solution was filtered through a 0.45 µm
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filter to obtain the operationally defined dissolved organic carbon (DOC) [Bianchi,
2011]. All stock solutions were prepare to yield a final concentration of approximately
1000 µM-C added DOC, after 1 mL of the stock solutions were added to 30 mL of
artificial seawater (ASW), which was buffered to a pH of 8.11 [Berges et al., 2001],
for the halogenation experiment.
Though using high purity salts for making the ASW, it has an average DOC
concentration of 285 µM-C, probably due to organic contaminants sorbing to the
hydrated salts used in the seawater recipe. Therefore, a relatively high DOC
concentration was needed to suppress the influence of background DOC present in the
ASW. Moreover, a high concentration of model DOM compound was used to ensure
that the DOM did not become a limiting factor for BrVSLS production. Actual DOC
concentrations for each model compound in ASW were determined with a Shimadzu
TOC-VCSH/CSN analyzer using high-temperature catalytic oxidation (HTC), as
described by Guo et al. [1994].
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Table 5-1. List of model DOM compounds used in this study with their chemical formula, with final DOC concentrations (µM) after added to artificial seawater (ASW), and summary of BrVSLS production properties. “N/A” indicates bulk macromolecule without a defined chemical formula. “Interfered” notes DOM that yielded significantly less BrVSLS than V-BrPO treated ASW. “No observable effect” notes addition of DOM did not enhance or interfere with BrVSLS production, relative to V-BrPO treated ASW. “Enhanced” notes DOM that is capable of producing significantly more BrVSLS than V-BrPO treated ASW. It should be note that DOM that interfered with BrVSLS production is not due to inhibition of V-BrPO.
Chemical formula Measured DOC concentration (µM)
BrVSLS production properties
Blank seawaters Artificial seawater (ASW) N/A 285.13 BrVSLS produced
Aged Gulf of Mexico seawater (GoMSW) N/A 171.00 Enhanced Seawater with model DOM added
Tryptophan C11H12N2O2 1702.50 Interfered Tyrosine C9H11NO3 1650.83 Interfered
Ferulic Acid C10H10O4 1488.33 Interfered Phenol Red C19H14O5S 1691.67 Interfered Thiamine C12H17N4OS 831.17 Interfered Vanillin C8H8O3 1640.83 Interfered Lignin N/A 734.75 Interfered
Syringaldehyde C9H10O4 1950.83 Interfered Glutamic Acid C5H9NO4 1663.33 Interfered Ascorbic Acid C6H8O6 1455 Interfered Aspartic Acid C4H7NO4 1570.83 Interfered
D-Glucose C6H12O6 1759.58 No observable effect Glucuronic Acid C6H10O7 819.58 No observable effect
Galactose C6H12O6 1575.00 No observable effect Xylose C5H10O5 1606.67 No observable effect
Maltoheptaose C42H72O36 979.17 No observable effect Starch (C6H10O5)n 1347.50 No observable effect
Pyruvic Acid C3H4O3 1205.83 No observable effect Maltose C12H22O11 1240.83 No observable effect
Methanol CH4O 1367.50 No observable effect Acetone C3H6O 903.33 No observable effect
Vitamin B12 C63H88CoN14O14P 1480.00 No observable effect Mannose C6H12O6 1247.50 No observable effect
Glycolic Acid C2H4O3 1485.83 Enhanced Alginic Acid (C6H8O6)n 771.25 Enhanced Citric Acid C6H8O7 1264.17 Enhanced Humic Acid N/A 324.42* Enhanced*
Urea CH4N2O 1771.11 Enhanced
* Flags low DOC concentration due to difficulty in completely dissolving the compounds into ultra-pure water.
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5.2.2 Preparation of V-BrPO stocks
V-BrPO extracted from a red calcifying macroalga, Corallina officinalis, was
purchased from Sigma-Aldrich®. The enzyme powder was dissolved in 50 mM MES
buffer at pH 6.4 and subsequently diluted to an appropriate concentration, such that
delivering 10 µL of V-BrPO stock to 30 mL of ASW yielded a final enzyme
concentration of ~ 0.07 units mL-1 for the experiment. One unit V-BrPO is defined as
the catalytic conversion of 1.0 µM of monochlorodimedon (MCD) to
monobromochlorodimedon per min at pH 6.4 at 25°C [Rush et al., 1995]. The
conversion of MCD to its brominated product was monitored with a Shimadzu UV
mini 1240 model UV-vis spectrophotometer, at 290 nm in disposable UV cuvettes
(applicable range λ = 220 to 900 nm, 1 mL sample volume, 1 cm path width). The
assay instruction was provided by Sigma-Aldrich®, which is based on Rush et al.
[1995]. Four different batches of enzyme were used during the experiments.
5.2.3 Halogenation reaction – single DOM model compound
Halogenation reaction mediated by V-BrPO was conducted in a 100 mL
ground glass gas tight syringe, with 30 mL of ASW, 1 mL of model DOM compound
stock (final concentration ~1000 µM-C), 10 µL V-BrPO (final concentration 0.07
units mL-1), and 10 µL H2O2 (final concentration 5.0 µM). All components were added
to the syringe without headspace and without bubbles. The addition of H2O2 activated
the reaction. The reaction fluid was mixed by gently inversing the syringe with two
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small pre-combusted glass beads inside to facilitate mixing. The reaction was
conducted in dark at 24.1 °C in a temperature controlled incubator for 4 hours. All
experiments were conducted in triplicate.
Six control sets were run to determine influences of different reaction
components on BrVSLS production: (1) blank ASW was run for each of the three
batches made during the experiment (ASW blank); (2) only H2O2 added to ASW with
model DOM compounds, which was run at the beginning of the experiment (H2O2
control); (3) only V-BrPO added to ASW with model DOM compounds, which also
was run at the beginning of the experiment (V-BrPO control); (4) only model DOM
compounds added to ASW, which was run for each model compounds (DOM control);
(5) V-BrPO mediated halogenation of ASW without model DOM compound added,
which was run whenever a new batch of ASW and/or V-BrPO was used (HASW).
Therefore, final BrVSLS concentrations yielded from halogenation of each model
DOM compound were compared to BrVSLS yielded from halogenation of the same
batch of ASW and enzyme.
5.2.4 Halogenation reaction – dual DOM model compounds
To better understand how DOM dynamics and interactions in the natural
environment may influence BrVSLS production, an over simplified DOM pool was
created by mixing two model DOM compounds. A model DOM compound that
interfered with BrVSLS production was mixed with a model DOM compound that
enhanced BrVSLS production in the following proportions: 50%, 10%, 0.5%, and 0%
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of DOM that interfered with BrVSLS production in a total added DOM concentration
of 1000 µM-C. A model DOM compound that did not affect BrVSLS production was
also mixed with a model DOM compound that enhanced BrVSLS production in the
same proportion as aforementioned, to compare BrVSLS production with the
aforementioned experiment. The DOM model compounds selected for this experiment
were based on results obtained from the single model DOM compound experiments.
5.2.5 BrVSLS analytical method
Samples for BrVSLS analysis were immediately transferred to a purge-and-
trap sample storage module after 4 hours of halogenation reaction. Sample replicates
run at different times in 8 hours after the reaction yield consistent BrVSLS
concentrations, which indicated BrVSLS production was completed and stable at the
time for analysis. HOBrenz reaction with DOM is a rapid process, which occurred on
the order of seconds to a maximum of 1 hour. Therefore, the long analytical time (~40
min per sample) did not affect BrVSLS quantification.
The purge-and-trap sample storage module is a compact thermoelectric
refrigerator held at ~ 24 ºC for this particular experiment. Inside, seven ~10 mL
calibrated volume glass bulbs were connected to a loop selection valve (Valco
Instrument Co.), which was connected to the purge-and-trap system. This
configuration allowed the samples to be stored in a constant temperature, in dark, and
gas tight environment until injection to the analytical system [Yvon-Lewis et al.,
2004]. Each sample was gently injected into the appropriate bulb through a 0.45µm
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cellulose acetate syringe filter. BrVSLS samples with and without passing through the
filters were compared and showed consistent results, which indicated the cellulose
acetate syringe filter did not affect BrVSLS quantification. All samples were analyzed
within 8 hours from the end of reaction.
For BrVSLS analysis, each sample in the glass bulb was sequentially
transferred into a glass sparger with an ultra-pure nitrogen gas stream, and purged at
144 mL min-1 at room temperature. The whole sample line was then flushed with the
same ultra pure nitrogen gas stream until it was time for the next sample to ensure no
residue from the previous sample was left in the line. The sample purge stream was
dried with two in-line Nafion driers (Perma Pure, NJ), and the analytes were pre-
concentrated on one cryotrap and then focused on a second cryotrap that were both
held at -75°C, and then desorbed at 210ºC. The analytes were desorbed from the
focusing trap and transferred by the same stream of ultra-pure nitrogen transfer gas
into the gas chromatograph – mass spectrometer (GC-MS) for quantification (Agilent
GC 6890N and MS 5973N; DB-VRX 0.240 ID column with 15m pre-column and 45m
main column). Detailed analytical methods were described in Yvon-Lewis et al. [2004]
and references therein.
Moist whole air gas calibration standards used in the experiment were
calibrated against gas standards obtained from the National Oceanic and Atmospheric
Administration Global Monitoring Division (NOAA GMD). The experiment
calibration standard was run after every fourth injection. The ultra-pure nitrogen gas
stream was run as a blank after every three samples to monitor flushing efficiency.
The detection limits for CHBr3 and CH2Br2 were 1.0 × 10-3 pmol L-1, and 5.0 × 10-3
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pmol L-1 for CHClBr2 and CHBrCl2. Analytical uncertainty was determined by
multiple injections of the calibration standard. The averaged analytical uncertainty was
4.4% for CHBr3, 6.0% for CH2Br2 , 1.5% for CHClBr2, and 2.6% for CHBrCl2. Purge
efficiency was determined by re-stripping seawater samples three times, and the
percentage in total concentration for the first stripping was 85.2% for CHBr3, 89.0%
for CH2Br2, 96.1% for CHClBr2, and 98.7% for CHBrCl2. BrVSLS concentrations
reported here were corrected for purge efficiencies.
5.2.6 Data quality control and statistics
Dixon’s Q-test was used to determine significant outliers from a small number
of samples (n = 3) at a 90% confidence level. Data points rejected by the Dixon’s Q-
test were removed when interpreting the data. All data were combined and examined
the overall correlations between the BrVSLS. The combined data were checked for
normality. Since the combined data showed a non-normal distribution, Spearman
Rank correlation was used to determine association between the BrVSLS, at a 95%
confidence level. The significance of the correlation was determined using a two-
tailed test. Significant differences of means between data groups were determined
using the One-Way ANOVA test at a 95% confidence level. All statistical analyses
were done with the OriginLab© version 9.0 statistic module.
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5.3 Results
5.3.1 BrVSLS production by single DOM model compounds
Results from the control groups indicate that the DOM control, H2O2 control,
and V-BrPO control do not produce BrVSLS in ASW on their own. BrVSLS
concentrations observed from these control experiments were not significantly
different from the ASW blanks. These control groups and ASW blanks consistently
yield a mean concentration of 9.1 ± 5.4 pmol L-1, 7.6 ± 3.0 pmol L-1, and 3.8 ± 1.7
pmol L-1, for CHBr3, CHClBr3, and CHBrCl2, respectively. CH2Br2 was not detectable
in the controls. Variability in BrVSLS concentrations were mainly due to different
background trihalomethane (THM) concentrations in the ultra-pure water used to
make up the ASW. ASW itself is capable of producing significant amounts of
BrVSLS upon halogenation (i.e. HASW), with an average CHBr3 concentration
ranging from 845.7 ± 290.5 pmol L-1 to 2140.9 ± 273.6 pmol L-1 between treatments
by different batches of V-BrPO (Table 4-2).
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Table 5-2. BrVSLS concentrations measured after 4-hours reaction of V-BrPO treated artificial seawater (ASW), aged Gulf of Mexico seawater, and samples with model DOM compounds added to ASW.
Chemical formula BrVSLS Concentrations (pmol
L-1) CHBr3 CHClBr2 CHClBr2
Seawaters Artificial seawater 1 N/A 2140.9 ± 273.6 160.6 ± 61.1 44.7 ± 11.7 Artificial seawater 2 N/A 845.7 ± 290.5 59.1 ± 24.0 22.4 ± 10.7 Artificial seawater 3 N/A 954.5 ± 370.4 83.3 ± 26.6 23.4 ± 5.7 Artificial seawater 4 N/A 1861.6 ± 557.2 109.6 ± 29.7 39.2 ± 9.8
Aged Gulf of Mexico seawater N/A 3609.6 ± 267.2 241.0 ± 18.2 70.3 ± 1.8 Seawater with model DOM added Tryptophan C11H12N2O2 8.9 ± 0.6 12.5 ± 9.8 4.7 ± 3.3
Tyrosine C9H11NO3 23.7 ± 10.8 11.0 ± 1.8 4.0 ± 0.4 Ferulic Acid C10H10O4 18.4 ± 9.6 8.8 ± 1.7 3.5 ± 0.6 Phenol Red C19H14O5S 41.3 ± 21.4 10.1 ± 2.8 9.2 ± 5.9 Thiamine C12H17N4OS 25.1 ± 20.9 11.6 ± 0.3 6.9 ± 3.0 Vanillin C8H8O3 54.4 ± 11.8 20.8 ± 7.3 7.4 ± 2.4 Lignin N/A 139.8 ± 60.0 14.7 ± 0.2 5.0 ± 0.004
Syringaldehyde C9H10O4 66.8 ± 22.5 15.5 ± 4.3 5.4 ± 1.3 Glutamic Acid C5H9NO4 177.1 ± 94.2 24.6 ± 19.9 6.0 ± 0.4 Ascorbic Acid C6H8O6 109.3 ± 83.7 26.3 ± 8.5 16.4 ± 3.3 Aspartic Acid C4H7NO4 1359.0 ± 518.7 38.2 ± 27.4 10.7 ± 4.1
D-Glucose C6H12O6 373.0 ± 13.1 53.1 ± 8.5 26.0 ± 3.8 Glucuronic Acid C6H10O7 657.3 ± 28.7 46.1 ± 1.9 15.8 ± 4.5
Galactose C6H12O6 717.1 ± 495.3 40.9 ± 22.6 12.8 ± 7.3 Xylose C5H10O5 759.2 ± 312.9 216.2 ± 141.9 24.6 ± 7.2
Maltoheptaose C42H72O36 1926.9 ± 8.9 183.3 ± 13.4 42.9 ± 5.1 Starch (C6H10O5)n 848.7 ± 325.4 60.1 ± 8.1 22.0 ± 5.0
Pyruvic Acid C3H4O3 921.3 ± 238.3 62.1 ± 18.7 13.6 ± 4.8 Maltose C12H22O11 1215.6 ± 456.7 89.4 ± 31.2 24.3 ± 7.3
Methanol CH4O 1214.7 ± 137.5 72.3 ± 10.2 23.1 ± 3.9 Acetone C3H6O 1633.9 ± 670.7 83.1 ± 25.1 31.1 ± 11.5
Vitamin B12 C63H88CoN14O14P 1151.9 ± 145.4 70.9 ± 4.4 23.1 ± 1.4 Mannose C6H12O6 1215.6 ± 456.7 89.4 ± 31.2 24.3 ± 7.3
Glycolic Acid C2H4O3 1646.8 ± 499.9 199.4 ± 100.0 33.3 ± 6.8 Alginic Acid (C6H8O6)n 1689.7 ± 185.3 140.5 ± 20.9 59.2 ± 4.1 Citric Acid C6H8O7 5511.2 ± 590.7 562.7 ± 3.0 104.4 ± 22.6 Humic Acid N/A 2633.8 ± 171.6 202.9 ± 12.1 83.9 ± 12.0
Urea CH4N2O 6980.1 ± 1748.2 203.3 ± 50.0 56.2 ± 6.9
Based on final BrVSLS concentrations observed at the end of the 4-hours
reaction with model DOM compounds added, the model DOM compounds were
categorized into three groups based on their BrVSLS production properties: (1)
enhanced BrVSLS production (DOMenhanced), (2) no observable effect on BrVSLS
production (DOMno_effect), and (3) interfered with BrVSLS production (DOMinterfered)
(Table 5-1, Figure 5-1). Enhanced BrVSLS production indicates the observed
concentrations were significantly higher than their corresponding batch of ASW
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treated with the corresponding batch of V-BrPO. No observable effect on BrVSLS
production indicates the observed concentrations were not significantly different from
their corresponding batch of ASW treated with the corresponding batch of V-BrPO.
Interfered with BrVSLS production indicates the observed concentrations were
significantly lower than their corresponding batch of ASW treated with the
corresponding batch of V-BrPO.
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Figure 5-1. BrVSLS concentrations of (a) CHBr3, (b) CHClBr2, and (c) CHBrCl2, measured from HOBrenz halogenation of a series of model DOM compounds. Grey bar chart shows BrVSLS concentration resulted from different batches of ASW treated with different batches of V-BrPO. Filled circles are mean concentrations of BrVSLS produced by each individual model DOM compound, with error bar of ± 1 standard deviation, plotting on top of their corresponding batch of ASW halogenated by their corresponding batch of V-BrPO. DOM group labeled red indicates BrVSLS production was significantly less than V-BrPO treated ASW without model DOM added (interfered). DOM group labeled black indicates BrVSLS production was not significantly different from V-BrPO treated ASW without model DOM added (no observable effect). DOM group labeled green indicated BrVSLS production was significantly higher than V-BrPO treated ASW without model DOM added (enhanced).
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A total of 11 out of 28 DOM model compounds significantly interfered with
BrVSLS production in ASW. Tryptophan, tyrosine, and ferulic acid effectively
interfered with BrVSLS production, such that the observed BrVSLS concentrations
were not significantly different from the ASW blanks. Only 5 out of 8 model DOM
compounds were capable of enhancing BrVSLS production, which included glycolic
acid, alginic acid, citric acid, humic acid, and urea. It should be noted that humic acid
was presented at a much lower concentration in the ASW than the rest of model DOM
compounds, since only a small fraction of it can be dissolved in ultra-pure water
(Table 5-1). The rest of the model DOM compounds showed no observable effects on
BrVSLS production in seawater. With all the data in this experiment combined, these
BrVSLS showed a significant overall correlation with each other (Table 5-3, Figure 5-
2). CH2Br2 production was not observed across all DOM model compounds tested.
Table 5-3. Spearman rank correlation coefficient (ρ) between the BrVSLS (p-value; number of samples n).
CHClBr2 CHBrCl2 V-BrPO halogenation Single substrate experiment
CHBr3 0.94
(<< 0.001; 80) 0.85
(<< 0.001; 78)
CHClBr2 - 0.92 (<< 0.001; 78)
Mix substrate experiment CHBr3
0.94 (<< 0.001; 48)
0.83 (<< 0.001; 48)
CHClBr2 - 0.89 (<< 0.001; 48)
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Figure 5-2. Scatter plot of CHBr3 concentration vs. CHClBr2 and CHBrCl2 concentrations for (a) single DOM model compound experiment and (b) dual DOM model compounds experiment. Blue data points presented CHBr3 concentration vs. CHClBr2 concentration. Red data points presented CHBr3 concentration vs. CHBrCl2 concentration. In panel (b), Filled squares are data observed from tryptophan mixed with urea. Filled triangles are data observed from vanillin mixed with urea. Stars are data observed from phenol red mixed with urea. Open up-side-down triangles are data observed from D-glucose mixed with urea. Open circles are data observed from ultra-pure water mixed with urea.
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Figure 5-3. Brominated trihalomethane speciation across the model DOM compounds examined, and in comparison with pre- and post- V-BrPO treated artificial seawater (ASW and HASW, respectively) and aged Gulf of Mexico seawater (GoMSW and HGoMSW, respectively).
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CHBr3, CHBrCl2, and CHClBr2, are also known as brominated
trihalomethanes (BTHM). Although significantly correlated with each other, the
relative abundances of these compounds varied significantly as a function of DOM
type (Figure 5-3). Relative abundances of CHClBr2 and CHBrCl2 tend to be higher in
samples with DOMinterfered added. CHClBr2 and CHBrCl2 relative abundances were
also higher in non-V-BrPO treated artificial seawater and aged Gulf of Mexico
seawater (GoMSW). Upon V-BrPO induced halogenation, CHBr3 relative abundances
significantly increased in ASW and GoMSW.
5.3.2 BrVSLS production by dual DOM model compounds
Three model DOMinterfered compounds, tryptophan, vanillin, and phenol red,
were chosen for the dual DOM experiment. Each compound was mixed at ratios
defined in section 5.2.4 with urea, a DOMenhanced. In addition, D-glucose, which
belongs to the DOMno_effect group, and ultra-pure water were individually mixed with
urea.
As shown in Figure 5-4, mixtures containing tryptophan, vanillin, and phenol
red at the relative proportions of 50%, 10%, and 0.5% of the added DOM
concentration resulted in significant reductions in BrVSLS concentrations, relative to
0% addition of these compounds (i.e. 100% added urea). CHBr3 concentration trends
between these treatments were consistent, as determined by one-way ANOVA test. D-
glucose mixed with urea in various proportions exhibited a similar trend as ultra-pure
water mixed with urea, as determined with a one-way ANOVA test. Such a
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consistency between D-glucose and ultra-pure water mix experiments confirmed that
the CHBr3 production trends observed from these experiments were resulted from
dilution of urea, and D-glucose does not affect CHBr3 production.
CHClBr2 and CHBrCl2 concentration trends observed from all dual model
DOM compounds experiments were similar to CHBr3. With all the data in the mixing
experiments combined, these BrVSLS exhibited a significant overall correlation with
each other, which suggested CHClBr2 and CHBrCl2 production responded to DOM
dynamics in a similar fashion as CHBr3 (Table 5-3 and Figure 5-2). BTHM relative
abundances also changed across different mixing proportion of DOMinterfered (Figure 5-
5). 50% and 10% relative proportion of DOMinterfered yield significantly less relative
abundances of CHBr3 than those observed from 0.5% and 0% relative proportion of
DOMinterfered.
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Figure 5-4. CHBr3 concentrations observed from dual model DOM compound experiment. (a) Tryptophan, (b) vanillin, (c) phenol red, (d) D-glucose, and (e) ultra-pure water were mixed with urea at relative proportions of 50%, 10%, 0.5%, and 0% (i.e. 100% urea), to make up a total DOM concentration of ~1000 µM. Grey bar chart plots CHBr3 concentration observed from V-BrPO treated ASW.
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Figure 5-5. Brominated trihalomethane speciation at mixing proportion of 50%, 10%, 0.5%, and 0% of DOMinterfered and DOMno_effect with urea: (a) Tryptophan, (b) vanillin, (c) phenol red, (d) D-glucose, and (e) ultra-pure water.
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5.4 Discussion
5.4.1 BrVSLS production sensitivity to DOM type
Our results from V-BrPO induced HOBrenz halogenation of single DOM model
compounds show a significant effect of DOM type on BrVSLS production and
speciation (Figures 5-1 and 5-3), when V-BrPO and H2O2 were present. The majority
of the model DOM compounds examined did not express observable effect on
BrVSLS production, relative to V-BrPO treated ASW. All carbohydrates examined in
this study expressed such a BrVSLS production pattern. Carbohydrate is one of the
dominant extracellular DOM produced by phytoplankton and encompasses a
significant fraction of the DOM in the ocean [Aluwihare and Repeta, 1999; Aluwihare
et al., 1997; Hansell et al., 2002]. In the surface ocean, carbohydrate can account for
as much as 50% of the bulk DOM [Aluwihare et al., 1997; Benner et al., 1992;
McCarthy et al., 1996]. It is not surprising that such a ubiquitous pool of DOM does
not have a significant effect on BrVSLS production, as BrVSLS only presented at
trace level on a global scale in a patchy distribution pattern.
The rest of the model DOM compounds either interfered with or enhanced
BrVSLS production, with more of them interfering with BrVSLS production.
Interestingly, amino acids and DOM compounds with phenolic ring structures, which
have been shown to produce considerable amounts of BrVSLS during drinking water
chlorination processes at alkaline pH (e.g. pH 8) [Hong et al., 2009; Reckhow et al.,
1990], interfered with BrVSLS production relative to HASW. Drinking water
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chlorination is analogous to V-BrPO induced halogenation [Quack and Wallace, 2003;
Ram et al., 1990; Wever and Van der Horst, 2013]. Therefore, we substituted V-BrPO
and H2O2 with NaOCl, an active ingredient in commercial bleach, to confirm whether
these compounds competitively inhibited the enzyme. These compounds also
interfered with BrVSLS production via NaOCl induced halogenation relative to
NaOCl treated ASW, which confirmed that the reduction in BrVSLS production was
not caused by competitive inhibition of the enzyme. However, unlike drinking water
treatment, amino acids in ASW interfered with BrVSLS production, which may
suggest different halogenation kinetics for these compounds in the seawater matrix,
with relatively low concentration of NaOCl. Amino acids and lignin phenols are
important DOM fraction present in the coastal seawater [Bianchi, 2011; Duan and
Bianchi, 2007], where significant amounts of BrVSLS being produced and served as
an important source of these ODSs to the atmosphere [Carpenter et al., 2009; Liu et
al., 2011; Liu et al., 2013; Quack and Wallace, 2003]. Therefore, their interference
with BrVSLS production in seawater can potentially influence coastal BrVSLS
dynamics.
Although only a few of the DOM compounds examined in this study enhanced
BrVSLS production relative to HASW, most of these compounds are important
metabolites produced by phytoplankton. Glycolic acid was one of the DOM
compounds that slightly enhanced BrVSLS production upon V-BrPO induced
halogenation. This particular metabolite is produced during photorespiration by
phytoplankton [Leboulanger et al., 1997; Leboulanger et al., 1998a; Leboulanger et
al., 1998b]. H2O2, which is required for V-BrPO to generate HOBrenz, is also produced
129
in light via photolysis of DOM and phytoplankton photosynthesis [Cooper et al.,
1988; Manley, 2002]. A strong correlation between H2O2 and CHBr3 concentrations
were observed from several species of phytoplankton isolated from the Baltic Sea
under photo-oxidative stress [Abrahamsson et al., 2003]. Such a condition is favorable
for CHBr3 formation, and now we show that it is not only because more H2O2 is
available for HOBrenz formation, but it is also owing to the fact that some metabolites,
such as glycolic acid, can enhance CHBr3 production during photorespiration.
Urea, an important metabolite produced by organisms, showed significant
enhancement of BrVSLS production. A substantially elevated CHBr3 concentration
was observed near the Canary Islands with low chlorophyll a concentration
[Carpenter et al., 2009; Liu et al., Submitted]. Elevated heterotrophic bacteria
abundance and urea concentration were also observed in this area [Liu et al.,
Submitted]. Liu et al. [Submitted] speculated that the presence of reactive DOM led to
elevated BrVSLS concentrations in this area, and the results presented in this study
support this hypothesis. Urea is an indicator for amino acid cycling by bacteria
[Remsen, 1971; Zehr and Ward, 2002], and it is also one of the DOM types that is
effective for BrVSLS production. In addition, bacteria can excrete and utilize urea
[Zehr and Ward, 2002] and many other DOM molecules. Since bacteria play an
important role in regulating DOM composition in seawater, they may play an
important, though maybe indirect, role in BrVSLS production.
Alginic Acid, a polysaccharide that is widely distributed in macroalgae
[McLachlan, 1985], also enhanced BrVSLS production. It is well known that brown
algae are prolific producers of BrVSLS [Carpenter and Liss, 2000]. V-BrPO activity
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was observed in a diverse group of macroalgae [Manley, 2002; Manley and Barbero,
2001; Theiler et al., 1978; Wever and Van der Horst, 2013; Wever et al., 1991; Wever
et al., 1993]. The prolific BrVSLS production from macroalgae has long been
explained by the presence of abundant V-BrPO. However, it was shown that CHBr3
production was reduced when the macroalgae (Ulva lactuca) was exposed to common
macroalgal metabolites [Manley and Barbero, 2001]. Therefore, the presence of V-
BrPO does not necessarily lead to enhanced BrVSLS production, unless a suitable
DOM is present, such as alginic acid.
Contrary to the phenolic DOM that interfered with BrVSLS production, humic
acid was able to substantially enhance BrVSLS production at low concentration,
which is consistent with results reported by drinking water treatment studies [Oliver
and Thurman, 1983; Reckhow et al., 1990]. Humic acid has been shown as a reactive
substrate for BrVSLS formation upon halogenation with HOBr, which formed either
via an enzyme-mediated pathway or NaOCl [Lin, 2011; Oliver and Thurman, 1983;
Reckhow et al., 1990]. This finding suggests only a small amount of reactive DOM is
required to have a significant impact on BrVSLS production, which is consistent with
observation by Lin and Manley [2012]. The authors observed a higher BrVSLS
production by halogenating high molecular weight (HMW) DOM. Although the
relative DOC concentration was lower in the HMW fraction, it was capable of
producing comparable amounts of BrVSLS as the low molecular weight (LMW)
DOM, which comprised a larger fraction of the bulk DOC. However, I speculate that
the presence of reactive functional groups is more important than DOM molecular size
alone. Besides the phenolic components, humic acids are also enriched in carboxylic
131
components, which are known to be reactive THM precursors [Boyce and Hornig,
1983].
It should be noted that CH2Br2 production was not observed in any of the
treatments. This result is also consistent with Lin and Manley [2012], who found that
only trace amount of CH2Br2 was produced upon V-BrPO induced halogenation of
seawater collected from certain locations in a certain season at the California coast.
Based on results from field observations, Liu et al. [2013] suggested CH2Br2 and
CHBr3 may not be derived from common sources (i.e. the same enzymatic mediated
path). The co-production of these two BrVSLS may be controlled by different
biogeochemical factors within the same ecosystem [Liu et al., 2013]. The same
conclusion was drawn from large BrVSLS datasets presented in the Atlantic Ocean
[Liu et al., Submitted]. A recent laboratory study by Hughes et al. [2013] suggested
that CH2Br2 was formed via biological mediated transformation of CHBr3. Our results
add further evidence for such a hypothesis, as CH2Br2 was not produced upon
completion of V-BrPO induced halogenation reaction, and phytoplankton or bacteria
cells were not present to facilitate the biological mediated transformation as proposed
by Hughes et al. [2013]. However, the possibility of CH2Br2 production being more
selective in DOM types cannot be excluded. Nonetheless, these would still suggest
that CH2Br2 is not always co-produce with CHBr3 via the same enzyme-mediated
BrVSLS production pathway.
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5.4.2 BrVSLS production in a dynamic DOM environment
The dual model DOM compounds experiment conducted in this study only
presented a highly simplified scenario compared with in situ. Nonetheless, it is clear
that BrVSLS production is govern by complex processes, such as the interaction
between different DOM molecules. The presences of DOMinterfered, even at trace
relative concentrations (0.5%), can substantially reduce BrVSLS production.
Ultimately, BrVSLS production is driven by the relative concentrations of different
compounds within the complex DOM pool in seawater and their reactivity.
DOM that is susceptible to V-BrPO induced halogenation does not necessarily
lead to BrVSLS production. The interference of BrVSLS production observed in this
study may be due to the fact that those molecules are more susceptible to
halogenation, but they formed halogenated non-volatile compounds instead of
producing BrVSLS. For example, aspartic acid and amino acids with similar
properties may form haloacetic acid (HAA) upon halogenation [Hong et al., 2009].
Phenol red is known to be a reactive DOM for halogenation, and it was used in a
HOBrenz assay [Hill and Manley, 2009; Sandy et al., 2011; Wever et al., 1991]. Phenol
red is yellow in acidic condition (pH ≤ 6) and red in alkaline condition (pH ≥ 8). Upon
bromination by HOBr, it turns into bromophenol blue, which is blue. Such a color
change upon bromination can be observed with a spectrophotometer. I followed the
transformation of phenol red to its brominated product. I found that while phenol red
significantly reduced BrVSLS production when the it was mixed with urea, it also
transformed into bromophenol blue (Figure 5-6). At trace relative concentration
133
(0.5%), phenol red also substantially reduced BrVSLS production, which suggests
phenol red is probably more susceptible to halogenation than urea.
These findings are consistent with Manley and Barbero [2001] who also found
that addition of phenol red reduced CHBr3 production. The halogenation of phenol red
occurs at its phenol rings; it is therefore reasonable to assume that lignin phenols
behave similarly. In fact, phenols are known antioxidants [Vinson et al., 2001], which
means they can react readily with oxidants like HOBr. Therefore, although not
producing volatile compounds, it is not surprising that these molecules are reactive in
forming other products, such as the non-volatiles, upon reaction with HOBr. Along
this line, although not containing phenolic structure, ascorbic acid (vitamin C) is also a
DOMinterfered and an antioxidant. Therefore, chemical properties of DOM and relative
abundances of different DOM types govern the halogenated products, such as the
relative abundance of halogenated volatile and non-volatile compounds. This will
ultimately govern the amounts of BrVSLS being produced in seawater.
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Figure 5-6. Absorbance of phenol red (λ = 433) and bromophenol blue (λ = 592) before (initial) and after (final) V-BrPO treatment of 10% phenol red + 90% urea samples.
5.4.3 Influence of DOM on brominated trihalomethane speciation
Our results showed that the relative abundance of CHBr3, CHClBr2, and
CHBrCl2 in the total BTHM pool is also sensitive to DOM type (Figure 5-5).
Interestingly, the relative abundance of the BTHM in non-V-BrPO treated ASW and
GoMSW was similar to those observed from the V-BrPO treated DOMinterfered. I
postulate that the DOMinterfered are kinetically more favorable in forming halogenated
135
non-volatile compounds rather than forming volatile compounds like BrVSLS. The
BTHM pools in the V-BrPO treated DOMinterfered are, therefore, remained unaltered as
the untreated seawaters samples.
The dual model DOM compounds mixing experiments also showed different
BTHM relative abundances. As considerable amounts of DOMinterfered (i.e. 50% and
10%) mixed with urea, they yielded a different BTHM relative abundance pattern
(Figure 5-5). This finding supports the above hypothesis. As the DOMinterfered are
kinetically more favorable for halogenation than urea, but formed halogenated non-
volatile molecules instead. Therefore, the BTHM pools in the 50% and 10% mixed
experiments were relatively less altered than those observed in the 0.5% and 0%
relative proportion of DOMinterfered. Hence, CHBr3 relative abundances in the 50% and
10% mixed experiments were lower, and more similar to the untreated ASW. The
effect of DOMinterfered in BTHM speciation was smaller at relative proportion of 0.5%.
Therefore, BTHM relative abundances in the 0.5% mixed experiments expressed
similar patterns as in the D-glucose and ultra-pure water mix experiments and the
100% urea (i.e. 0% DOMinterfered) experiment.
In a dynamic environment, such as the ocean, the relative abundances of
DOMinterfered in the bulk DOM pool may lead to different patterns of BTHM relative
abundance, as observed in the Atlantic Ocean (Figure 5-7).
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Figure 5-7. Brominated trihalomethane speciation observed during the Halocarbon Air-sea Transect – Atlantic (HalocAST-A) cruise.
5.4.4 Implications for BrVSLS biogeochemistry in the ocean
CHBr3 concentration in seawater follows a broad trend with chlorophyll a
concentration [Carpenter et al., 2009; Liu et al., Submitted; Schall et al., 1997]. A
clear concentration gradient of CHBr3 across waters with different ranges of
productivity was observed in the Atlantic Ocean [Liu et al., Submitted]. In the water
137
column, the BrVSLS tend to be elevated near the subsurface chlorophyll maximum
depth [Liu et al., 2013; Quack et al., 2004]. These observations show that the
involvement of phytoplankton, and presumably V-BrPO activity, is essential for
BrVSLS production. However, BrVSLS concentration usually does not significantly
correlate with chlorophyll a concentration. Even though BrVSLS tend to be elevated
with chlorophyll a, significant BrVSLS variability in productive waters was observed.
Several explanations have been suggested, including effects of sea-to-air fluxes of
BrVSLS, different turnover rates between BrVSLS and chlorophyll a, phytoplankton
species specificity for BrVSLS production, physical mixing, influence of
anthropogenic input, and DOM specificity for BrVSLS production [Abrahamsson et
al., 2004; Hughes et al., 2009; Liu et al., 2011; Liu et al., Submitted; Mattson et al.,
2012].
While these processes can, to some extent, alter the relationship between
BrVSLS and chlorophyll a, a factor that is more directly linked to the enzyme-
mediated path was suggested, namely the variability in V-BrPO activity in seawater
[Wever and Van der Horst, 2013]. However, only a small fraction of HOBrenz
ultimately turns into CHBr3, as observed by Hill and Manley [2009], which suggests
V-BrPO induced HOBrenz may not be a limiting factor for BrVSLS production in
seawater. Results from this study offer another plausible explanation on the significant
variability of BrVSLS concentration in seawater. I hypothesize that the variability in
BrVSLS production is affected by DOM type in seawater. As aforementioned, the
relative abundance and reactivity of DOMinterfered and DOMenhance will lead to different
BrVSLS production rates in seawater.
138
The coastal ocean is a significant source of BrVSLS [Carpenter et al., 2009;
Liu et al., 2011; Quack and Wallace, 2003; Quack et al., 2007a], owing to its high
productivity and presence of macroalgal habitats. Moreover, reactive DOM is also
present at high concentrations in the coastal ocean. However, DOMinterfered, such as
tryptophan, tyrosine, and lignin phenols can also occur in the coastal ocean. These
compounds also cycle quickly in along the freshwater-to-saline water gradient [Coble,
2007; Hernes and Benner, 2003]. Therefore, the fate of different types of DOM will
ultimately govern what is available for HOBrenz halogenation and the subsequent
release BrVSLS.
Hence, to better understand BrVSLS biogeochemistry, it is important to better
understand DOM characterization. CDOM comprise an important fraction of the
DOM [Coble, 2007]. Characterization of CDOM with excitation-emission matrix
couple with parallel factor analysis (PARAFC) is becoming an emerging tool to
understand components in the CDOM pool. Such a method is relatively economical
and can be easily deployed in the field. Therefore, it is possible to utilize information
in DOM characteristic in BrVSLS modeling study; once we have more information on
the relationship between DOM characteristic and BrVSLS production.
5.5 Conclusion
Our results suggest the type of DOM in seawater can significantly influence
BrVSLS production, which has important implications for BrVSLS concentrations,
distributions, and emissions from the ocean. These factors will ultimately affect the
139
global BrVSLS budget and supply to the stratosphere via SGI. Moreover, in a future
climate, DOM supply to the coastal ocean and DOM fate may experience drastic
changes. For example, as the artic permafrost starts thawing and more labile DOM is
discharged to the coastal ocean. Such changes will likely shift relative abundances of
DOM type that can influence BrVSLS, hence, significantly alter the production of
BrVSLS in coastal waters. Since coastal oceans are known as “hot-spots” for the
BrVSLS, such changes may have significant impacts on global BrVSLS budgets.
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CHAPTER VI
PRODUCTION OF BROMINATED VERY SHORT-LIVED SUBSTANCES
(BRVSLS) BY PHYTOPLANKTON
6.1 Introduction
Brominated very short-lived substances (BrVSLS) are important inorganic
bromine (Bry) suppliers to the atmosphere. Bromoform (CHBr3) and dibromomethane
(CH2Br2) account for ~80% of the very short-lived organic bromine in the marine
boundary layer [Law and Sturges, 2007], and these two major BrVSLS account for
~76% of very short-lived substances (VSLS) derived Bry (BryVSLS) in the stratosphere
[Hossaini et al., 2012a]. Chlorodibromomethane (CHClBr2) and
bromodichloromethane (CHBrCl2) comprise a relatively minor fraction of the
BrVSLS, and they are capable of supplying moderate amount of BryVSLS to the
stratosphere [Hossaini et al., 2012a]. These BrVSLS degraded into Bry via photolysis
and reaction with hydroxyl radicals (OH). The resulting Bry can then participate in
catalytic ozone destruction cycles. On an atom-by-atom basis, bromine is ~50 to 100
times more effective in destroying ozone than chlorine [Garcia and Solomon, 1994;
Solomon et al., 1995]. Therefore, numerous efforts have been made to better
understand BrVSLS global budgets, distributions, and sources [Butler et al., 2007;
Carpenter and Liss, 2000; Carpenter et al., 2009; Carpenter et al., 2007b; Liu et al.,
2011; Liu et al., 2013; Liu et al., Submitted; Moore and Tokarczyk, 1993; Moore et
141
al., 1996; Quack and Wallace, 2003; Quack et al., 2007a; Quack et al., 2007b; Quack
et al., 2004].
CHBr3, CH2Br2, CHClBr2, and CHBrCl2 are mainly derived from natural
sources in seawater. Macroalgae are prolific producers of natural BrVSLS, and a wide
range of macroalgal species produce substantial amounts of BrVSLS [Carpenter and
Liss, 2000; Gschwend and MacFarlane, 1986]. However, despite the large BrVSLS
production strengths, macroalgal habitats are constrained to certain types of coastal
environments [Lüning, 1990]. Therefore, phytoplankton is hypothesized to be a
globally significant source, owing to their ubiquitous distribution in the surface ocean
[Moore et al., 1995b]. Several groups of phytoplankton are known to produce BrVSLS
in laboratory cultures, including diatoms and coccolithophores [Colomb et al., 2008;
Hughes et al., 2013; Moore et al., 1996]. However, owing to substantially elevated
BrVSLS concentrations were observed in the Arctic and Antarctic oceans, most
studies had focused on cold-water phytoplankton species.
Here, I report results observed from screening of 9 phytoplankton species for
BrVSLS production.
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6.2 Methods
6.2.1 Phytoplankton culture
I selected 9 phytoplankton species from diverse classes, including a few
tropical and subtropical species (Table 6-1; Figure 6-1). 8 of the phytoplankton species
chosen for this screening study are cosmopolitan. Selection of phytoplankton was
based on (1) correlation between BrVSLS and biogeochemical markers observed from
field studies, which cosmopolitan species of the taxonomic groups found to be
correlated with BrVSLS were selected; (2) cosmopolitan diatoms that collected from
temperate and warm waters; (3) phytoplankton species that were known to express V-
BrPO activity.
Phytoplankton stock cultures were purchased from the National Center for
Marine Algae and Microbiota (NCMA; formerly known as Culture Collection of
Marine Phytoplankton, CCMP). All phytoplankton cultures were grown in 2 L
incubation vessels with ~ 600 mL culture medium inside (Figure 6-2). 0.2 µm filtered
artificial seawater (ASW) [Beggs et al., 2009] was added to each culture vessel and
autoclaved for 40 min and allowed to cool prior to nutrient and culture additions.
Nutrients were added to each vessel at concentrations suggested by NCMA. For most
phytoplankton cultures, L1 nutrients were used [Guillard and Hargraves, 1993].
Cyanobacteria cultures were grown in L1 media without silicate added. The
crytophyte, Guillardia theta, was grown in K nutrients, which is designed for
oligotrophic oceanic species [Keller et al., 1987]. Finally, 1 mL of culture stock was
143
added to each vessel. Once inoculated, the culture vessel entrance were flamed and
capped. All cultures were grown in triplicate.
Two incubation vessels with only ASW and nutrients added were incubated
along with the cultures as blanks. All procedures were conducted in a LABCONCO
vertical clean bench to avoid bacterial contaminations. All cultures were incubated in a
temperature-controlled incubator at 20 °C in a 10/14 hr-light/dark cycle. Photon flux
density (pfd) near the incubation vessels surfaces were ~42 µmol-photon m-2 s-1. Trace
gases in the headspaces were analyzed for each sample on the day of inoculation with
gas chromatography – mass spectrometer (GC-MS). All cultures were then closed and
incubated until they reached stationary phase to accumulate sufficient amounts trace
gases for production measurement. Cell growth phase was estimated by light
transmission through each culture vessel as described below. It should be noted that
Achnanthes longipes was grown in both ASW and Gulf of Mexico seawater
(GoMSW) to compare whether BrVSLS production was different in natural seawater.
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Figure 6-1. Map of locations where the stock cultures were isolated from. Information was provided by the National Center of Marine Algae and Microbiota (NCMA). Orange symbols indicate diatom species, magenta symbol indicates cryptophte species, purple symbol indicates dinoflagellate species, grey symbol indicates prasinophyte species, green symbol indicates open ocean cyanobacteria species, and pink symbol indicates coastal cyanobacteria species. Stars indicate species with BrVSLS production.
145
Table 6-1. Phytoplankton species screened for BrVSLS and CHCl3 production. “+” indicates significant production of BrVSLS and CHCl3 were observed in the culture. “-” indicates there were no observable production of BrVSLS and CHCl3 in the culture. “c.d.” notes production cannot be determined due to co-elution of unidentified substances with the compounds of interested.
Species CCMP ID# Class Medium used CHBr
3 CH2Br
2 CHClBr
2 CHBrCl
2 CHCl
3
Achnanthes longipes 101 Bacillariophyceae L1, ASW and GoMSW + + + + +
Synechococcus sp. 2515 Cyanophyceae L1 - Si, ASW - + + + + Odontella aurita 1796 Coscinodiscophyceae L1, ASW c.d. c.d. c.d. c.d. c.d. Phaeodactylum
tricornutum 2561 Bacillariophyceae L1, ASW - - - - -
Thalassiosira weissflogii 1051 Coscinodiscophyceae L1, ASW - - - - - Ostreococcus sp. 2972 Prasinophyceae L1, ASW - - - - - Synechococcus elongatus_cf 1379 Cyanophyceae L1 - Si, ASW - - - - -
Guillardia theta 327 Cryptophyceae K, ASW - - - - - Karenia brevis 2228 Dinophyceae L1 - Si, ASW - - - - -
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Figure 6-2. Schematic of incubation vessel used for phytoplankton culture screening. Any trace gases produced by the phytoplankton were equilibrated with the headspace. Port A was connected to the GC-MS system to analyze for halocarbon production. A 0.2µm air filter was connected to port B, which served as makeup gas inflow to compensate pressure change inside the vessel during halocarbon headspace sampling.
A B
Culture Medium
Headspace
Makeup gas inflow tube
Gas sampling port Makeup gas inflow tube
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6.2.2 Assess cell density in culture
Due to the nature of trace gas sampling, the incubation vessels cannot be
opened to draw samples for cell counts as the cultures grow. Therefore, growth in each
vessel was semi-qualitatively assessed by measuring the amount of light transmitted
through the culture vessel every day. Each culture vessel was rolled gently on a rolling
machine and then placed in front of a constant full spectrum light source. A LiCor LI-
250A light meter was placed in line with the light source on the other side of the
culture vessel to measure light transmission through the culture vessel (Figures 6-3
and 6-4). The measurement of light transmission was conducted in dark to avoid
ambient light interfering with the light source. Cell density is presented as a function
of light transmission, which is calculated with the following equation:
Itransmission = Iculture – Iblank (Equation 6.1)
where Itransmission is the difference of light intensity transmitted through the
culture and blank. The more negative the value, the higher the cell density in the
vessel. Pfd was measured in µmol-photon m-2 s-1.
148
Figure 6-3. Schematic of light transmission method, used for assessing cell density. A full spectrum light source was place at one side of the incubation vessel. A LiCor LI-250A light meter was place inline on the other side of the incubation vessel to measure light transmission through the incubation vessel.
A B
Full spectrum light source
Gas sampling port Makeup gas inflow tube
Light meter
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Figure 6-4. An example of cell density (Achnanthes longipes Agardh), presented in light transmission (Itransmission, µmol-photon m-2 s-1), as a function of time. Trace gas samples were analyzed once the cell cultures reached stationary phase.
150
6.2.3 Trace gas analysis
Trace gas concentrations were measured on a cryo-trapping and focusing –
GC-MS system, as described by [Yvon-Lewis et al., 2004]. Each sample was gently
rolled on a rolling machine for 2 hours to achieve equilibrium in the headspace, and
then connected (port A, refer to Figure 6-2) to a 12-port automated stream select valve
in connection with a vacuumed sampling line. A 0.2 µm membrane filter was
connected to port B (refer to Figure 6-2) to ensure no bacterial contamination was
introduced into the vessel when the make-up air was drawn into the vessel. Make up
air was ambient room air drawn into the vessel during sample collection to
compensate change in headspace pressure inside of the vessel. Approximately 1 L of
headspace was drawn from the headspace of each incubation vessel. Pressure of the air
collection canister was recorded for calculating the actual headspace volume drawn as
trace gas sample. Subsequently, the valve was turned into the next position with an
ultra-pure nitrogen stream connected. The ultra-pure nitrogen stream served as make-
up gas for the trace gas analysis. Analytes were trapped and focused at -80 °C and
desorbed and injected into the GC column at 210 °C.
A moist air standard, calibrated against a whole air working-standard obtained
from the National Oceanic and Atmospheric Administration (NOAA) Global
Monitoring Division (GMD), was used as a calibration standard. Calibration air was
run at the beginning of a sequence and after every forth injection as: calibration,
sample 1, sample 2, sample 3, ultra-pure nitrogen, and calibration. Ultra-pure nitrogen
151
stream was run after every third injection of sample to monitor the make-up gas
quality, and ensure no contamination in the sampling line.
Detection limits (in pico atmosphere, patm) for CHBr3, CH2Br2, CHBrCl2,
CHClBr2, and CHCl3 were 0.02 patm, 0.03 patm, 0.01 patm, 0.01patm, and 0.46 patm,
respectively. Average analytical uncertainty was 7.0% for CHBr3, 6.0% for CH2Br2,
9.0% for CHBrCl2 and CHClBr2, and 6.7% for CHCl3. All trace gas concentrations
reported here were converted to aqueous concentrations (C, pmol L-1) using the
following equation:
C = !!
(Equation 6.2)
where p is the equilibrated partial pressure of the gas of interested in patm, and
H is the Henry’s Law constant. For the BrVSLS, H was converted from the
dimensionless Henry’s law constant reported by Moore et al. [1995a], using the gas
constant (0.0821 L atm K-1 mol-1). Similarly, H for chloroform (CHCl3) was calculated
from the dimensionless Henry’s law constant reported by Dewulf et al. [1995].
6.2.4 Statistics
Significant outlier was determined by Dixon’s Q-test at a confidence level of
90%. Data points rejected by the Dixon’s Q-test, if any, were removed when
interpreting the data. Amounts of trace gases produced over the course of incubation
were determined by the concentration differences between those observed on the day
of inoculation and those observed at stationary phase. One-way ANOVA test at a
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confidence level of 95% was used to determine whether the production was significant
(i.e. significantly different from day 1).
6.3 Results
Cell growths were observed in all cultures, but only 2 out of 9 phytoplankton
species screened showed observable production of BrVSLS (Table 6-1). A. longipes
and Synechococcus sp. were capable of producing significant amounts of various
BrVSLS over the course of incubation (Table 6-2, Figure 6-5). Significant production
of CHBr3, CH2Br2, CHClBr2, CHBrCl2, and CHCl3 were observed in A. longipes
cultures. Significant amounts of CHBr3, CHClBr2, and CHBrCl2 were observed in
Synechococcus sp. cultures. However this particular strain of Synechococcus did not
produce observable amounts of CH2Br2 and CHCl3.
BrVSLS concentrations and chemical speciation differ substantially between
these two species. Importantly, BrVSLS concentrations and chemical speciation also
differ significantly from the same species, A. longipes, which incubated under similar
conditions but in different seawater sources and incubation lengths. Two batches of A.
longipes were grown in ASW for 30 days and 11 days, respectively. One batch of A.
longipes was grown in open Gulf of Mexico seawater (GoMSW) for 11 days.
Although the same class of BrVSLS was produced in these batches, their relative
abundances were different (Figures 6-6 and 6-7). CHBr3, CHClBr2, and CHBrCl2 are
brominated trihalomethanes (BTHM). The BTHM and CHCl3 are trihalomethanes
(THM). CHBr3 relative abundances in the BTHM pool differ substantially in the three
153
batches of A. longipes cultures (Figure 6-6). CHBr3 was consistently a minor
production within the THM pool (Figure 6-7) in A. longipes cultures. In
Synechococcus sp. culture, CHBr3 was a major production within the BTHM pool,
with a pattern similar to those observed in typical enzymatic-mediated bromination
reactions [Liu et al., In prep] (see Chapter V) (Figure 6-6).
Table 6-2. Concentrations (±1 standard deviation) of CHBr3, CH2Br2, CHClBr2, CHBrCl2, and CHCl3 produced in A. longipes and Synechococcus sp. cultures.
Species (condition) CHBr3 (pmol L-1)
CH2Br2
(pmol L-1) CHClBr2
(pmol L-1) CHBrCl2
(pmol L-1) CHCl3
(pmol L-1) A. longipes
(L1, ASW, incubated for 30 days) 179.2 ± 46.7 9.1 ± 1.0 23.3 ± 7.5 117.0 ± 25.6 1821.6 ± 529.1
A. longipes (L1, ASW, incubated for 11 days) 1507.1 ± 244.8 121.2 ± 13.8 1025.6 ± 145.7 3649.8 ± 46.6 472.6 ± 0.7
A. longipes (L1, GoMSW, incubated for 11 days) 268.9 ± 79.4 471.4 ± 58.0 601.1 ± 70.3 2190.6 ± 220.9 944.7 ± 86.6
Synechococcus sp. (L1 - Si, ASW, incubated for 14 days) 1331.8 ± 694.9 - 302.3 ± 167.6 78.5 ± 48.8 -
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Figure 6-5. Amounts (in aqueous concentration pmol L-1) of CHBr3, CH2Br2, CHClBr2, CHBrCl2, and CHCl3 produced by A. longipes and Synechococcus sp. in cultures of different ages. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater.
155
Figure 6-6. Relative abundances of CHBr3, CHClBr2, and CHBrCl2 in the brominated trihalomethane (BTHM) pool in A. longipes (A.I.) and Synechococcus sp. (Syn) cultures. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater.
156
Figure 6-7. Relative abundances of CHBr3, CHClBr2, CHBrCl2, and CHCl3 in the trihalomethane (THM) pool in three different batches of A. longipes (A.I.) cultures. ASW indicates that the cultures were grown in artificial seawater, and GoMSW indicates that the culture was grown in Gulf of Mexico seawater.
157
6.4 Discussion
6.4.1 Is BrVSLS production species specific?
Results observed in this study indicate that the production of BrVSLS is highly
species specific, which is consistent with findings from other studies [Colomb et al.,
2008; Hill and Manley, 2009; Hughes et al., 2013; Moore et al., 1996; Moore et al.,
1995b; Tokarczyk and Moore, 1994]. Results in this study also support numerous field
observations, which found that BrVSLS concentrations do not significantly correlate
with chlorophyll a and other pigment biomarkers; and species-specificity has long
been an explanation [Abrahamsson et al., 2004; Ekdahl et al., 1998; Liu et al., 2013;
Mattson et al., 2012]. However, recently, Liu et al. [Submitted] found that, on a basin-
wide scale, elevated BrVSLS concentrations in the ocean were often located in regions
with high concentrations of chlorophyll a. A clear gradient of CHBr3 concentration
was observed across waters with different production (estimated with chlorophyll a
concentration). This observation by Liu et al. [Submitted] not only confirmed the
importance of phytoplankton in mediating BrVSLS production, but also indicates that
phytoplankton species that are capable of mediating BrVSLS production are more
widespread than have long been believed.
Results presented by Liu et al. [In prep] (see Chapter V) provided new insights
to these discrepancies. The two species that were capable of producing BrVSLS, A.
longipes and Synechococcus sp., have been shown to express bromoperoxidase
activity [Hill and Manley, 2009; Johnson et al., 2011]. Vanadium bromoperoxidase
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(V-BrPO) is an enzyme involved in mediating halogenation of dissolved organic
matter (DOM) and the subsequent release of BrVSLS in seawater [Butler and Carter-
Franklin, 2004; Lin and Manley, 2012; Theiler et al., 1978; Wever et al., 1993].
Therefore, results in this study are consistent with the BrVSLS formation pathway
proposed over a decade ago. However, the role of DOM has recently been suggested
to play an essential role in controlling BrVSLS production [Liu et al., In prep] (see
Chapter V). Therefore, the apparent species specificity in BrVSLS production may
have been influenced by DOM composition in the culture medium.
Liu et al. [In prep] (see Chapter V) has shown that 2 out of 3 vitamins
commonly used in phytoplankton culture, namely, thiamine and ascorbic acid (vitamin
C), interfered with V-BrPO mediated BrVSLS production. Common amino acids
produced by phytoplankton, such as aspartic acid and glutamic acid [Granum et al.,
2002], also significantly interfered with V-BrPO mediated BrVSLS production. The
author also suggested that these compounds formed non-volatile halogenated
molecules instead of the volatile compounds. Therefore, species that only express
weak V-BrPO production may not yield observable BrVSLS production, as these
DOM molecules tend to be present at high concentration in a phytoplankton culture.
Moreover, if the DOM molecules produced by certain phytoplankton species are more
reactive in forming non-volatile molecules, then BrVSLS may not be observed.
As suggested by Liu et al. [In prep] (see Chapter V), BrVSLS production
strength may ultimately control by the DOM pool in the environment. Some
compounds enhance BrVSLS production while some interfere with BrVSLS
production. Therefore, DOM present in the phytoplankton culture may significantly
159
alter BrVSLS production rate. The extracellular DOM produced by a phytoplankton
cell is complicated and involves many factors, such as cell physiological state, any
potential chemical reactions occur in the medium, environmental factors (e.g.
temperature and light), and presence of bacteria. As discussed by Kujawinski [2010],
DOM profile observed from monocultures can be drastically different from cultures of
multiple species. This indicates interactions between different phytoplankton species
and the surrounding microbial community can significantly change the DOM pool,
hence, the fate of BrVSLS production.
It has been also shown that the presence and absence of bacteria can alter
CHBr3 production [Hughes et al., 2013]. I proposed two plausible explanations. First,
the composition of the DOM produced by bacteria is different from what is produced
by phytoplankton. It is possible that the change in DOM characteristic with the
presence of bacteria changes BrVSLS production. Second, V-BrPO has been proposed
as a defense mechanism for phytoplankton [Manley, 2002; Ohsawa et al., 2001; Wever
et al., 1991]. Halogenated molecules, created via V-BrPO mediated halogenation,
have been shown to disrupt bacterial quorum sensing [Sandy et al., 2011]. Therefore,
the presence of bacteria may trigger more V-BrPO halogenation activity and alter
BrVSLS production. However, controlling bacterial abundance in cultures with
antibiotic may be problematic as well, as these molecules may interfere with BrVSLS
production.
The ocean in reality is so much more complicated than a monoculture.
Considering the complexity of DOM, results from laboratory monoculture cannot be
used to understand BrVSLS biogeochemistry in the ocean. To legitimately determine
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whether BrVSLS production is indeed species specific, independent of DOM
influence, more information on V-BrPO activity in culture is required. Unfortunately,
V-BrPO assay is usually not a routine exercise for BrVSLS production screening
studies.
6.4.2 A different production path for BrVSLS in A. longipes?
As discussed by Liu et al. [In prep] (see Chapter V), DOM type can
significantly influence the relative abundances of BTHM. Different patterns in the
relative proportions of BTHM produced in different batches of A. longipes cultures
may be an indicator of shifts in DOM composition. CHBr3 was consistently a major
product among the BTHM in Synechococcus sp. and experiments with V-BrPO treated
DOM compounds, following the pattern of CHBr3 > CHClBr2 > CHBrCl2 (Figure 6-5
and 6-6). However, CHBr3 was only a minor product in A. longipes cultures.
Chloroform (CHCl3) is usually not considered in V-BrPO mediated reactions;
however, CHCl3 and/or CHBrCl2 consistently constituted a large fraction of THM
produced in A. longipes cultures. CHCl3 was a dominant product in batch of A.
longipes cultures incubated for 30 days. Although not as substantial, CHCl3 was
produced at a considerable level in the other two batches of A. longipes cultures
incubated for 11 days (Figure 6-5 and 6-7). In these two batches, the other chlorinated
THM, CHBrCl2, was the dominant product. It should be noted that CHCl3 was not
produced in the Synechococcus sp. culture, in which BTHM relative production
pattern followed a typical V-BrPO mediated bromination product pattern.
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The first explanation of such a shift in product relative abundances in A.
longipes cultures could be successive chlorine substitution from CHBr3, following
such an order: CHBr3 à CHClBr2 à CHBrCl2. However, such a process is believed
to be a long time scale process, occurring on the order of years to decades [Geen,
1992]. Such a pattern was observed in the Pacific Ocean in aged North Pacific
Intermediate Water (NPIW), with an apparent CFC-11 age of a few decades [Liu et
al., 2013]. Therefore, while successive chlorine substitution may be one of the
explanations, it would be too slow to explain the high CHCl3 concentrations observed
in all A. longipes cultures.
The second explanation could be enzyme-mediated chlorination, basically
similar to V-BrPO bromination. Vanadium or iron-heme chloroperoxidases are widely
distributed in terrestrial environments, where they are involved in the degradation of
lignin [Ortiz-Bermudez et al., 2003; Ortiz-Bermúdez et al., 2007]. Unlike
bromoperoxidase, which can only catalyze the oxidation of bromine and iodine,
chloroperoxidase can catalyze the oxidation of chlorine, bromine, and iodine. For a
long time, the marine environment is thought to only host two haloperoxidases,
bromoperoxidase and iodoperoxidase. However, it has been shown that V-BrPO from
a brown alga can express chloroperoxidase-like activity, i.e. catalyze the oxidation of
chlorine [Soedjak and Butler, 1990; Winter and Moore, 2009]. Therefore, it is possible
that certain phytoplankton, such as A. longipes can also express chloroperoxidase-like
activity. Such a finding will have important implications in developing our
understandings of natural chlorinated trace gas budgets. Moreover, chloroperoxidase-
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like activity offers a simple explanation for the observation of elevated CHCl3
concentration in the chlorophyll a maximum [Liu et al., 2013].
6.5 Conclusion
The original objective of this study was to screen important phytoplankton
species for the ability to produce BrVSLS, with the aim of elucidating which taxa are
potentially important producers in situ and to facilitate explaining the patchy
distribution of BrVSLS production in the ocean. However, screening phytoplankton
for BrVSLS production may not be as useful as it has been long believed. Although it
is typical that laboratory experiments tend to be different from in situ, the complex
factors involved in BrVSLS production make it even more so. BrVSLS production
seems to be maintained by interactions with a complex DOM pool, hence, traditional
approaches of growing phytoplankton cultures may be problematic. Moreover, to
screen for phytoplankton that can in reality produce BrVSLS, it is probably more
helpful to screen for V-BrPO activity rather than the BrVSLS themselves. More
attention should be paid to understand BrVSLS production pathways and environment
factors that can alter their production. Finally, I demonstrated for the first time,
chloroperoxidase-like behavior from a diatom.
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CHAPTER VII
EFFECTS OF VANADIUM BROMOPEROXIDASE HALOGENATION ON
DISSOLVED ORGANIC MATTER AND IMPLICATIONS FOR THE
MARINE CARBON CYCLE
7.1 Introduction
In the marine environment, halogenated non-volatile organic compounds such
as indoles, terpenes, acetogenins, phenols, and volatile organic compounds such as
some of the brominated very short-lived substances (BrVSLS) are thought to be the
result of halogenation reactions catalyzed by bromoperoxidase [Butler and Carter-
Franklin, 2004]. Thus far, two major classes of bromoperoxidase are known in the
marine environment: iron-heme bromoperoxidase and vanadium bromoperoxidase (V-
BrPO); with the vanadium dependent type being more abundant [Butler and Carter-
Franklin, 2004].
V-BrPO activities have been identified in many macroalgae, as well as several
groups of phytoplankton, such as diatoms and cyanobacteria [Deboer et al., 1986;
Gribble, 2010; Hill and Manley, 2009; Johnson et al., 2011; Moore et al., 1996;
Wever et al., 1991; Wever et al., 1993]. Although the exact physiological function of
this class of enzymes is not clear, it has been suggested to serve two purposes:
scavenging of oxidants like hydrogen peroxide (H2O2) and producing defensive acids
like hypobromous acid (HOBrenz) [Manley and Barbero, 2001; Wever, 2012]. HOBr is
a halogenating/oxidizing agent that can act on a wide range of organic substrates.
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HOBr is also a well-known mild acid that can transform aldose sugars into aldonic
acids, which can potentially alter carbohydrate characteristics as well as carbohydrate
bioavailability within the bulk dissolved organic matter (DOM) pool [Varela, 2003].
The halogenation of organic molecules, mediated by V-BrPO, starts with the
catalytic oxidation of halides X (for bromoperoxidase X = Br- and I-) in the presence
of H2O2, yielding oxidized halogen intermediates like HOBrenz. The resulting HOBrenz
can then react with suitable organic reactants to form halogenated organic molecules
[Theiler et al., 1978; Wever et al., 1993]. Under certain conditions, volatile organic
compounds such as CHBr3, CHClBr2, and CHBrCl2 can be formed from further
halogenation, with eventual cleavage from the macromolecule [Theiler et al., 1978;
Wever et al., 1993]. Since bromine is at much higher concentration than iodine in
natural seawater, the brominated products are dominant.
The idea of brominated very short-lived substances (BrVSLS) are formed via
V-BrPO mediated halogenation of DOM, is a widely accepted hypothesis. However,
the role of such a reaction pathway in driving global variability of BrVSLS seawater
concentrations has been largely neglected. Liu et al. [In prep] (see Chapter V)
demonstrated that BrVSLS production, hence their concentrations in seawater, varied
significantly across different model DOM compounds treated with V-BrPO. This
finding not only revealed a significant driver of BrVSLS global variability, but also
has significant implications for the carbon cycle. Upon halogenation, DOM molecules
were transformed either into unstable halogenated compounds and released BrVSLS
or into stable non-volatile halogenated compounds. These non-volatile halogenated
165
molecules may express different chemical reactivity and bioavailability, hence, will
have an impact on the carbon cycle.
Colored dissolved organic matter (CDOM) is a fraction of the bulk dissolved
organic matter (DOM) pool that absorbs light in both visible and ultraviolet (UV)
ranges [Blough and Del Vecchio, 2002]. In coastal waters, where riverine input is
significant, CDOM is primarily derived from terrestrial sources [Blough and Del
Vecchio, 2002]. This pool of DOM is important even though it is only a small fraction
of the bulk DOM pool, as its optical properties can significantly influence aquatic and
marine biogeochemistry. Liu et al. [In prep] (see Chapter V) demonstrated that certain
DOM compounds interfere with BrVSLS production and transform into non-volatile
products upon halogenation. Here, I used change in CDOM fluorescence to assess
transformation of CDOM upon treatment with V-BrPO, a process recently termed
‘bio-bleaching’. Bio-bleaching of CDOM was first hypothesized by Lin and Manley
[2012]; it is the enzymatic-mediated degradation of CDOM as oppose to photo-
bleaching. Specifically, the Lin and Manley [2012] referred to V-BrPO mediated
oxidation of CDOM.
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7.2 Method
7.2.1 Preparation of colored dissolved organic matter material
Two dissolved organic matter (DOM) pools, resembling characteristics of
terrestrial- (allochthonous) and marine- (autochthonous) derived CDOM, were
prepared. Lignin phenols, including vanillin, syringaldehyde, and ferulic acid, were
dissolved in 0.2 µm filtered ultra pure water as stock solutions. 1 mL of stock solution
was added to 30 mL of artificial seawater (ASW; buffered to a pH of 8.11 and 0.2 µm
filtered) [Berges et al., 2001; Liu et al., In prep]. Final DOC concentrations were ~
1000 µM-C in the DOM added ASW. Syringaldehyde and ferulic acid were dissolved
in sub-boiling ultra-pure water. These lignin phenols were used as model
allochthonous CDOM, as they comprise a significant fraction of the DOM pool in
coastal seawaters [Bianchi, 2011; Hernes and Benner, 2003]. Moreover, the lignin
phenols have defined chemical structure so we could compare their reactivity. The
traditional CDOM model substances, such as humics and fulvics, were not used in this
study, as they have substantial differences in chemical composition and reactivity
depend on their sources.
Autochthonous CDOM was harvest from cultures of four phytoplankton
species, Achnanthes longipes (CCMP 101), Skeletonema costatum (CCMP 2092),
Synechococcus sp. (CCMP 2515), and Synechococcus elongates (CCMP 1379). The
selection of the cultures covered two important groups of phytoplankton, diatoms and
cyanobacteria. Aged cultures (i.e. at stationary or senescent phase) were gently filtered
167
through 0.2 µm filters to collect the operationally defined DOM [Bianchi, 2011]. It
should be noted that the CDOM collected with this method is qualitative and not
necessarily algal derived, as bacteria were present in the aged cultures and they may
have transformed the algal CDOM and/or produced CDOM. However, this will also
be true in the natural marine environment.
7.2.2 Preparation of V-BrPO and H2O2
V-BrPO stock was prepared as described in Liu et al. [In prep] (see Chapter
V). Basically, the enzyme powder was dissolved in 50 mM MES buffer (MES pH =
6.4). 4 µL of V-BrPO was added to a total volume of 108 µL reaction solution
(CDOM + H2O2 + V-BrPO). Final concentration of V-BrPO was ~13.7 units mL-1.
Hydrogen peroxide (H2O2) stock solution was made by diluting 30% v./v. H2O2 with
ultra-pure water. 4 µL of H2O2 was added to a total volume of 108 µL reaction
solution, with a final concentration of 0.56 mM. Concentrations of V-BrPO and H2O2
used in this study were two orders of magnitude higher than those used in Liu et al. [In
prep] (see Chapter V) in order to observe measureable bleaching of CDOM with a
microplate reader.
7.2.3 Fluorescence measurement
Fluorescence of CDOM was measured with a microplate reader (SpectraMAX
GEMINI EM) in top-read mode. A 96-well opaque microplate was used. Inside the
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plate, three controls of each CDOM were added. Controls were CDOM without V-
BrPO and H2O2. Samples for CDOM measurements were run in duplicates, due to
limited batch of V-BrPO. Basically, triplicates of CDOM were added to the
microplate. H2O2 were added to all samples and monitored for 5 min, to ensure
addition of H2O2 does not change CDOM fluorescence. Subsequently, V-BrPO was
added to two of the CDOM samples to activate halogenation. The remainder one
CDOM sample without V-BrPO was used to continue monitoring H2O2 induced
change. Halogenation reaction with each CDOM was run for at least 1.5 hr. The
microplate reader was set into kinetic mode, and made a fluorescence measurement
every 30 seconds. Relative CDOM concentration was measured using an excitation of
373 nm, emission of 460 nm, and auto optical emission cutoff light below 450 nm.
The microplate reader was maintained at a constant temperature of 25 °C.
7.3 Results
All CDOM samples with only H2O2 added showed the same trend as the
controls, which indicated potential CDOM oxidation by H2O2 did not change
fluorescence or optical properties of the CDOM types examined in this study. All the
fluorescence changes were stabilized after 1.5 hrs. A percent reduction in CDOM
fluorescence was calculated with the following equation:
%RFUreduced = RFUactived - RFUpre-activateRFUpre-activate
× 100% (Equation 7.1)
where RFU is the relative fluorescence unit. Activated denoted addition of V-BrPO to
activate halogenation. Pre-activate denoted addition of H2O2 only. % RFU offers a
169
qualitative measure of magnitude of bio-bleaching so that it is possible to compare
reactivity of a particular CDOM to V-BrPO induced halogenation.
All three lignin phenols showed signs of bio-bleaching upon halogenation by
V-BrPO induced HOBrenz (Figure 7-1). Vanillin, syringaldehyde, and ferulic acid
showed a reduction of RFU by 38.0%, 42.5%, and 65.5%, respectively. CDOM
collected from diatom cultures showed minor to no reduction in RFU (Figure 7-2). A.
longipes showed a reduction of RFU by 22.2%, while S. costatum showed no clear
trend in change of RFU. However, the RFU change in A. longipes was ambiguous as it
was not much different from the controls. CDOM collected from the two
cyanobacteria cultures showed a clear reduction in RFU. S. elongatus showed a RFU
reduction of 35.5%. Synechococcus sp. showed a RFU reduction of 21.7%. Although
such a RFU change from Synechococcus sp. was similar to that observed in A.
longipes, it was clearly different from the control (Figure 7-2). Therefore, I assume
alteration of A. longipes derived CDOM by V-BrPO induced HOBrenz was negligible.
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Figure 7-1. Fluorescence of V-BrPO treated (a) vanillin (red), (b) syringaldehyde (blue), and (c) ferulic acid (black) and their controls (grey) as a function of time. Error bars indicate 1 standard deviation (± 1σ) of replicates, number of samples (n) were 3 and 2 for control and V-BrPO treated lignin phenols, respectively.
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Figure 7-2. Fluorescence of V-BrPO treated CDOM collected from (a) Achnanthes longipes (orange), (b) Skeletonema costatum (purple), (c) Synechococcus elongates (green), and (d) Synechococcus sp. (pink) as a function of time. Fluorescence of their controls is presented in grey symbols. Error bars indicate 1 standard deviation (± 1σ) of replicates, numbers of samples (n) were 3 and 2 for control and V-BrPO treated CDOM collected from phytoplankton cultures, respectively. Note the different time scales of S. costatum and Synechococcus sp.
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7.4 Discussion
7.4.1 Bio-bleaching of CDOM
Here, I presented evidence of V-BrPO induced transformation of CDOM.
%RFUreduced for the lignin phenols exhibit such a pattern as: ferulic acid >
syringaldehyde > vanillin. This order is consistent with their relative
reactivity/stability in the environment [Dittmar and Lara, 2001; Tareq et al., 2004].
CDOM collected from cyanobacteria cultures appeared to be more reactive to V-BrPO
induced halogenation than CDOM collected from diatom cultures. This may be
important for CDOM biogeochemistry on a global scale, as cyanobacteria are
ubiquitous in the ocean [Waterbury et al., 1979].
CDOM, a fraction of DOM is of particular interest to researchers due to its
unique optical characteristics, which are important to processes that can affect
biogeochemistry and the ecological health of the aquatic environment. CDOM
strongly absorbs light in the visible and UV range, and thus plays a key role in
controlling light penetration in the water column, as well as the quantity and quality of
photosynthetic active radiation (PAR). The photoreactivity of CDOM leads to the
formation of a variety of compounds, which can then influence aquatic
biogeochemistry, carbon cycling, and atmospheric chemistry. These compounds
include low-molecular-weight OM that can be assimilated by prokaryotes [Benner and
Biddanda, 1998; Blough and Del Vecchio, 2002; Kieber et al., 1990; Mopper et al.,
1991; Moran et al., 2000; Zepp et al., 1998; Zepp et al., 2007], reactive species such
173
as H2O2 [Zafiriou et al., 1984; Zika et al., 1993], and different trace gases such as
carbon monoxide (CO), carbon dioxide (CO2), and carbonyl sulfide (COS) [Miller and
Zepp, 1995; Miller et al., 2002; Mopper et al., 1991; Stubbins et al., 2011; Stubbins et
al., 2008; Valentine and Zepp, 1993; Weiss et al., 1995; Zafiriou et al., 2003; Zepp et
al., 1998; Zepp et al., 2007].
Lignin degradation contributes in part, to the formation of humic and fulvic
acids, which comprise a large fraction of coastal CDOM, which has additional
contributions from amino acids, peptides, nucleic acids, and many other low-
molecular-weight OM [Nelson and Siegel, 2002]. CDOM absorption decreases
drastically along the freshwater to marine gradient. A similar pattern for lignin-
phenols was observed in surface waters from the lower river out through the
Mississippi River Plume (MRP) [Hernes and Benner, 2003], as CDOM fluorescence
in coastal waters usually correlated with lignin phenol contents [Boyle et al., 2009; Del
Vecchio and Blough, 2004]. Photo-oxidation (photo-bleaching) is the dominant
degradation path for CDOM in surface water. Bacterial degradation also contributes to
the loss of CDOM, but to lesser extent [Coble, 2007]. However, to our knowledge,
alteration of and/or “bleaching” of CDOM via phytoplankton biochemistry (e.g.
enzymes) has not been investigated in coastal waters.
Therefore, results in this study may provide new insights to CDOM
degradation. This is potentially important to regions where solar radiation is spatially
and temporally limited in the water column, such as the turbid coastal areas and polar
regions. Change in CDOM optical properties also implies chemical structural change.
A well-known paradigm of the terrestrially derived DOM, as the “lost” DOM from
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terrestrial sources, is that the annual DOC discharged to the global ocean can be
accounted for in the turnover time of all marine DOC; yet, various markers evidenced
that only small amount of terrestrially derived DOC ended up in the global ocean
[Bianchi, 2011]. Such a paradigm suggested the presence of effective loss
mechanism(s). Therefore, bio-bleaching may be an important pathway for OM
chemical alteration in addition to photo- and bacterial- oxidation, which may be an
important process in determining the fate of the “lost” terrestrial DOM in coastal
waters.
Besides direct transformation or degradation, compounds produced via bio-
bleaching may affect marine carbon cycling due to changes in bioavailability. HOBr
itself is a mild acid and it is known through fundamental organic chemistry to
transform carbohydrates into acids, for example, aldose sugars into aldonic acids
[Varela, 2003]. Amino acids are important DOM that cycle rapidly in the ocean as
they are utilized by microbes [Duan and Bianchi, 2007; Ittekkot and Zhang, 1989;
Spitzy and Ittekkot, 1991]. Aromatic amino acids, such as tyrosine and tryptophan, are
important CDOM constituents in the aquatic environment [Burdige et al., 2004;
Mopper and Schultz, 1993; Mopper et al., 1996]. These two compounds interfered
with BrVSLS formation and supposedly formed haloacetic acids (HAA) instead
[Hong et al., 2009]. HAAs are relatively stable in the marine environment [Hashimoto
et al., 1998]. Therefore, upon halogenation, these amino acids may be turned into
molecules that are not as bioavailable to the microbes. In fact, I hypothesize this may
be a general pattern for most of the halogenated molecules, as halogenated substances
tend to be toxic.
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7.4.2 Impacts beyond CDOM
While results presented in this study only demonstrated V-BrPO alteration of
CDOM, its impact on the bulk DOM pool is likely. Only a small fraction of HOBrenz
ultimately yielded BrVSLS [Hill and Manley, 2009; Lin and Manley, 2012].
Therefore, the fate of the remainder HOBrenz is open for speculation. Results presented
by Liu et al. [In prep] (see Chapter V) and this study clearly showed that HOBrenz has
significant impact on the non-volatile organic molecules. Bromine bounded organic
molecules were found in sinking particles throughout the water column, as well as in
sediments [Leri et al., 2010]. Therefore, halogenated organic compounds may be more
abundant than what we know thus far. Alteration in chemical properties of organic
matter (OM) due to halogenation may significantly influence OM degradation and
preservation in sediments, hence is important to diagenesis [Leri et al., 2010]. The OM
may be ‘stabilized’ upon halogenation, as its toxicity may protect it from bacterial
degradation in the sediment. V-BrPO is likely one of the important halogenation
pathway in the marine environment, though more halogenation pathways are yet to be
discovered.
7.5 Conclusion
In this study, I extended the scope of my research interests beyond trace gas
and BrVSLS biogeochemistry and examined the impact of V-BrPO mediated
halogenation on the carbon cycle in general. I demonstrated transformation of DOM
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via V-BrPO mediated halogenation, although the global significance of such a DOM
alteration pathway is still unknown. High concentrations of BrVSLS observed in the
Artic coast were attributed to high levels of V-BrPO activity. As the Artic permafrost
is starting to thaw and will discharge more labile DOM to the coast, V-BrPO mediated
halogenation may be one of the important pathway to alter this pool of reactive DOM.
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CHAPTER VIII
CONCLUSIONS
8.1 Result summary
Results from the field observations and laboratory experiments consistently
suggest that CHBr3 and CH2Br2 are derived from disparate sources. Better correlations
between CH2Br2 and the biological proxies observed in the field support the idea of
biologically mediated transformation of CH2Br2 from CHBr3 [Hughes et al., 2013].
The general correlation between CH2Br2 and CHBr3 suggest such a transformation
process is ubiquitous in the ocean, where CHBr3 is produced. Such a finding
challenges the long believed concept: CH2Br2 and CHBr3 are derived from common
sources. Such a contradiction may have important implications for modeling studies
on CH2Br2 and CHBr3 oceanic emissions and their global budgets, as various
environmental forcing may affect the two BrVSLS differently.
Results from the field observations generally indicate that the presences of
photosynthetic biomass are important for BrVSLS production. However, relationships
between the two are not straightforward. BrVSLS are believed to form via V-BrPO
mediated halogenation of DOM, in the presence of H2O2. To date, most of studies on
BrVSLS sources focus on phytoplankton specificity to BrVSLS production; i.e. in
relation to V-BrPO activity. Based on the field observations, I hypothesized that
BrVSLS production is controlled by complex factors in the ecosystems. Therefore,
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phytoplankton species specificity to BrVSLS or V-BrPO production is not sufficient
to explain BrVSLS production variability and biogeochemistry in the ocean.
Hence, I examined effects of extracellular DOM composition in controlling
BrVSLS production. Results from the laboratory studies show that CHBr3, CHClBr2,
and CHBrCl2 production is sensitive to DOM composition. Therefore, DOM
specificity to BrVSLS production may be a plausible explanation for the observed
variability in BrVSLS production, as DOM composition varied significantly in
different environments. Phytoplankton produces V-BrPO, which provides an initial
condition for halogenation to occur. However, as shown in the laboratory results,
DOM composition, reactivity, and dynamics control how much BrVSLS are
ultimately produced, assuming H2O2 is not a limiting factor.
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Figure 8-1. A conceptual model of BrVSLS production from within the phycosphere to the surrounding environment. 8.2 An outlook from a conceptual model
Based on the observed large-scale distributions of BrVSLS and laboratory
results, a conceptual model of BrVSLS is proposed. I propose that a BrVSLS
production “hot-spot” exists within the cell phycosphere (i.e. the micro-environment
around the cell) (Figure 8-1). V-BrPO has been proposed as an extracellular enzyme
bound to the cell surface [Manley, 2002]. Therefore, V-BrPO concentration is
presumably higher within the phycosphere. Metabolites that served as DOMenhanced,
such as urea, glycolic acid, and alginic acid, along with photorespiration by-product
such as H2O2 are at higher concentrations as they are being exuded by the cell. Such
an environment provides ideal conditions for BrVSLS formation. Although
180
DOMinterfered such as the amino acids will also exist at high concentrations in the
phycosphere, it is possible that enough of V-BrPO and DOMenhanced can counteract the
interferences. This conceptual model is supported by field observations that high
BrVSLS concentrations are usually located in biologically active regions of the ocean.
In the coastal ocean, reactive terrestrial DOM such as humics and fulvics may also
enhance BrVSLS production in the phycosphere. Beyond the phycosphere, excess
HOBrenz may diffuse further away and halogenate DOM in the surrounding waters.
Along such a path, HOBrenz may convert to bromine chloride (BrCl) [Sivey et al.,
2013]. Although HOBrenz may be present at a lower concentration further away from
the cell, it may encounter very reactive DOM in the coastal water, such as humic
substances. Although BrCl’s fate in seawater is elusive and it only occurs in minor
abundance compare to HOBr, it is believed to be a more effective brominating agent
than HOBr [Sivey et al., 2013]. Therefore, there are mechanisms for BrVSLS to form
away from the cell, although the magnitude will be highly dependent on fates of the
reactive bromine species in seawater and reactivity of DOM. The fact that BrVSLS
may be formed further away from the cell may be able to explain the complex
relationships between the BrVSLS and chlorophyll a.
It should also be noted that pH within the phycosphere can be drastically
different from the bulk medium, which is dependent on cell physiological state and
carbon chemistry in seawater [Dixon et al., 1989; Flynn et al., 2012; Kühn and
Köhler-Rink, 2008]. Such a pH difference from the surrounding environment may also
affect the amounts of BrVSLS being produced (Figure 8-2).
181
Figure 8-2. CHBr3 concentrations observed from halogenated artificial seawater (HASW) buffered to different pHs.
8.3 Conclusions and future works
To conclude, global distributions of BrVSLS are controlled by complex
environmental factors, such as those controlling HOBr fates in the ocean, as well as
those controlling DOM dynamics. My conceptual model derived from the results of
this dissertation suggests that most of the BrVSLS are produced in close proximity to
the cell, but phytoplankton species specificity is probably not as important as long
been believed. Moreover, a “life-after-death” concept was proposed by Manley and
182
colleagues (pers. comm. and Wever and Van der Horst [2013]). Basically, V-BrPO is
a stable enzyme that can survive after drastic temperature and pH changes. Since
HOBrenz produced via V-BrPO mediated processes is toxic, V-BrPO can potentially
protect itself from microbial degradation. Therefore, V-BrPO can remain active and
mediate halogenation even after cell death. Although this is merely a conceptual
model as well, it also highlighted that the important factors of halogenation reactions
are V-BrPO activity (i.e. HOBrenz supply) and DOM composition.
To better understand global BrVSLS distributions and the responses to climate
change, researchers should go beyond observations and start to understand
fundamental processes that control the production of BrVSLS. BrVSLS production
should be studied in conjunction with DOM composition and production mechanisms
of HOBr and/or V-BrPO in future studies. Identifying BrVSLS producers with
traditional biomarkers, such as pigments, is probably not useful, unless better tools at
molecular level are developed. Finally, although fully speculative, it is possible that
photochemistry and ozone deposition in seawater can create reactive brominated
species that can act as brominating agents. This may only be a minor pathway for
DOM bromination, but provided a mechanism that is completely independent of
phytoplankton.
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APPENDIX
Figure A-1. Schematic of the purge and trap analytical system, used for depth profile and V-BrPO treated DOM studies.
213
Figure A-2. Schematic of the stream select analytical system, used for continues air and equilibrator measurements and phytoplankton culture studies.